Hiv vaccine formulations

ABSTRACT

Provided herein are HIV vaccines comprising HIV polypeptide-encoding DNA adsorbed to PLG and/or HIV proteins. Also provided are methods of using these vaccines to generate immune responses in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of Ser. No. 10/530,543,filed Nov. 7, 2005, which is the U.S. National Phase of InternationalApplication No. PCT/US2003/031935, filed Oct. 7, 2003, which claims thebenefit of U.S. Provisional Application No. 60/416,573, filed Oct. 7,2002. The above applications are incorporated in their entirety byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by NIH HIVDDT GrantNo. N01-AI-05396 from the National Institutes of Health. The Governmenthas certain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to immunogenic HIV compositions,in particular to HIV vaccines and methods of formulating andadministering these vaccines.

BACKGROUND

Acquired immune deficiency syndrome (AIDS) is recognized as one of thegreatest health threats facing modem medicine. There is, as yet, no curefor this disease. In 1983-1984, three groups independently identifiedthe suspected etiological agent of AIDS. See, e.g., Barre-Sinoussi etal. (1983) Science 220:868-871; Montagnier et al., in Human T-CellLeukemia Viruses (Gallo, Essex & Gross, eds., 1984); Vilmer et al.(1984) The Lancet 1:753; Popovic et al. (1984) Science 224:497-500; Levyet al. (1984) Science 225:840-842. These isolates were variously calledlymphadenopathy-associated virus (LAV), human T-cell lymphotropic virustype III (HTLV-III), or AIDS-associated retrovirus (ARV). All of theseisolates are strains of the same virus, and were later collectivelynamed Human Immunodeficiency Virus (HIV). With the isolation of arelated AIDS-causing virus, the strains originally called HIV are nowtermed HIV-1 and the related virus is called HIV-2. See, e.g., Guyaderet al. (1987) Nature 326:662-669; Brun-Vezinet et al. (1986) Science233:343-346; Clavel et al. (1986) Nature 324:691-695. Since theimplementation of highly active antiretroviral therapy (HAART) in theUnited States in 1996, the number of persons diagnosed with acquiredimmunodeficiency syndrome (AIDS) and the number of deaths among personswith AIDS have declined substantially (Karon et al. (2001) Am J PublicHealth 91(7): 1060-1068) as a result, the number of persons living withAIDS has increased. The Centers for Disease Control (CDC) estimates thatas of Dec. 31, 2000, approximately 340,000 persons in the United Stateswere living with AIDS. (MMWR, Centers for Disease Control andPrevention. HIV/AIDS Surveillence Report, 13(No. 1) 2001).

Clinical trials in the US have been conducted with a limited number ofsubjects and further HIV vaccine development will require theidentification of a suitable population where the HIV seroincidence issufficiently high to enable a distinction between protection in theimmunized population with a placebo control. Seage III et al. (2001) Am.J. Epidemiol. 153(7):619-627; Halpern et al., (2001) J Acquir ImmuneDefic Syndr 27(3):281-8.

The primary mode of transmission of HIV is through sex and by contactwith infected body fluids including blood, semen, vaginal fluid, breastmilk, and other body fluids containing blood. In industrializedcountries, the majority of cases reported in which the person's risk isknown are among men who have sex with men. Before blood screening forantibodies to HIV was instituted, transfusion-associated HIV was aconcern in the US. (CDC. Update: HIV-2 infection among blood andplasmadonors—United States, June 1992-June 1995. MMWR, 1995. 44: p. 603-606).Other modes of transmission include needle sharing by injection drugusers, inadvertent contact with infected blood among hospital workers,and rare iatrogenic transmission through the re-use of contaminatedmedical equipment. Higher rates of sexually transmitted infectionssignal a rise in unsafe sex practices. Chen et al. (2001) Am J PublicHealth 92(9):1387-1388. Heterosexual transmission of HIV-1 continues torise, particularly among women, the young, and the economicallydisadvantaged and, in fact, heterosexual transmission is the dominantmode of transmission in the developing world. These trends highlight theneed for the development of a preventive and/or therapeutic vaccine.Catania et al. (2001) Am J Public Health 91(6):907-914.

Several targets for vaccine development have been examined, includingthe env and Gag gene products encoded by HIV. Gag gene products include,but are not limited to, Gag-polymerase (pol) and Gag-protease (prot).Env gene products include, but are not limited to, monomeric gp120polypeptides, oligomeric gp140 polypeptides (o-gp140) and gp160polypeptides.

Recently, use of HIV Env polypeptides in immunogenic compositions hasbeen described. (see, U.S. Pat. No. 5,846,546 to Hurwitz et al., issuedDec. 8, 1998, describing immunogenic compositions comprising a mixtureof at least four different recombinant virus that each express adifferent HIV env variant; and U.S. Pat. No. 5,840,313 to Vahlne et al.,issued Nov. 24, 1998, describing peptides which correspond to epitopesof the HIV-1 gp120 protein). In addition, U.S. Pat. No. 5,876,731 to Siaet al, issued Mar. 2, 1999 describes candidate vaccines against HIVcomprising an amino acid sequence of a T-cell epitope of Gag linkeddirectly to an amino acid sequence of a B-cell epitope of the V3 loopprotein of an HIV-1 isolate containing the sequence GPGR. However, thesegroups did not identify an effective HIV vaccine.

U.S. Pat. No. 6,602,705 and International Patent Publications WO00/39302; WO 02/04493; WO 00/39303; and WO 00/39304 describepolynucleotides encoding immunogenic HIV polypeptides from varioussubtypes.

Thus, there remains a need for immunogenic HIV compositions,specifically for HIV vaccine formulations.

SUMMARY

In one aspect, the invention includes an HIV DNA vaccine compositioncomprising a nucleic acid expression vector (e.g., plasmid, viralvector, etc.) comprising at least one HIV Gag- or Env-encoding sequenceand PLG. Preferably, the nucleic acid expression vector is adsorbed tothe PLG. In certain embodiments, the concentration of PLG is betweenabout 5 and 100 fold greater than the concentration of the nucleic acidexpression vector. For example, the concentration of nucleic acid can bebetween about 10 μg/mL and 5 mg/mL and the concentration of the PLG canbe between about 100 μg/mL and 100 mg/mL and/or the nucleic acidexpression vector concentration per dose can be between approximately 1μg/dose and 5 mg/dose and the PLG concentration per dose can be betweenapproximately 10 μg/dose and 100 mg/dose. Specific formulations aredescribed herein, for example, in Table 1, Table 2, or column 2 of Table9.

In another aspect, the invention includes an HIV vaccine compositioncomprising an HIV envelope protein, for example oligomeric gp140(o-gp140); and a pharmaceutically acceptable excipient. In certainembodiments, the concentration of o-gp140 is between about 0.1 mg/mL and10 mg/mL. Further, in certain embodiments, the concentration of o-gp140per dose is approximately 100 μg/dose. Specific formulations of HIVprotein vaccines are also described herein, for example in Table 3 andTable 11.

In another aspect, the invention comprises an HIV vaccine including oneor more of the HIV DNA vaccines described herein (e.g., an HIV Gag DNAvaccine as described herein and an HIV Env DNA vaccine as describedherein) and one or more of the HIV vaccines described herein (e.g., anHIV o-gp140 preparation).

Any of the HIV vaccine compositions described herein may further includeone or more adjuvants, for example MF59 or CpG. A particular formulationfor MF59 is set forth in Table 4.

In yet another aspect, the invention includes a method of generating animmune response in a subject, comprising (a) administering at least oneHIV vaccine composition described herein to the subject, and (b)administering, at a time subsequent to the administering of step (a), atleast one HIV vaccine composition described herein. In certainembodiments, the at least one HIV vaccine composition administered instep (a) comprises an HIV DNA vaccine (e.g., at least one HIV Gagvaccine and/or at least one HIV Env vaccine) as described herein and theHIV vaccine composition administered in step (b) comprises an HIVprotein vaccine as described herein. Furthermore, step (a) may comprisemultiple administrations of one or more HIV DNA vaccines as describedherein (e.g., two or three administrations at one month intervals) andstep (b) may comprise at least one administration of one or more HIVprotein vaccines as described herein (e.g., two or three administrationsat 1, 2, or 3 month intervals). Alternatively, step (b) may compriseconcurrently administering at least one HIV DNA vaccine described herein(e.g., an HIV Gag vaccine and/or an HIV Env vaccine) and at least oneand at least one HIV protein vaccine as described herein. The timebetween the administrations of step (a) and step (b) can vary, forexample between 1 to 6 months or even longer. In any of the methodsdescribed herein, one or more administrations may be intramuscularand/or intradermal.

In a further aspect, the invention includes a method of makingoligomeric HIV Env gp140 proteins, comprising the steps of introducing anucleic acid encoding gp140 into a host cell; culturing the host cellunder conditions such that gp140 is expressed in the cell; and isolatingoligomeric gp140 (o-gp140) protein from the host cell. In certainembodiments, the o-gp140 is secreted from the cell and isolated from thecell supernatant.

In a still further aspect, a method of making any of the HIV DNAvaccines described herein is provided. The method comprises the step ofcombining a nucleic acid expression vector comprising a sequenceencoding one or more HIV polypeptides with aseptic PLG microparticlessuch that the nucleic acid expression vector binds to the PLGmicroparticles to form a DNA/PLG HIV vaccine. In certain embodiments,the method further comprises the step of lyophilizing the DNA/PLG HIVvaccines.

In another aspect, the invention includes a method of making an HIVprotein vaccine as described herein, the method comprising the steps ofcombining o-gp140 with an adjuvant.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B are graphs depicting the effect of PLGmicroparticles on anti-Gag antibody responses induced by DNA vaccines.FIG. 1A shows geometric mean ELISA titers of animals immunized withplasmid DNA at weeks 0, 4 and 14, then boosted at weeks 38 and 75 withrecombinant Env protein formulated with MF59. FIG. 1B shows geometricmean titer of animals immunized with pSINCP DNA at weeks 0, 4 and 14,then boosted at weeks 38 and 75 with recombinant Env protein formulatedwith MF59. Anti-Gag antibodies are plotted as geometric mean ELISA titerfor naked pCMV (solid symbols) and PLG/pCMV (open symbols) and errorbars represent SEM.

FIG. 2A and FIG. 2B are graphs depicting the effect of PLGmicroparticles on anti-Env antibody responses induced by DNA vaccines.FIG. 2A shows geometric mean ELISA titers of animals immunized withplasmid DNA at weeks 0, 4 and 14, then boosted at weeks 38 and 75 withrecombinant Env protein formulated with MF59. FIG. 2B shows geometricmean titer of animals immunized with pSINCP DNA at weeks 0, 4 and 14,then boosted at weeks 38 and 75 with recombinant Env protein formulatedwith MF59. Anti-Env antibodies are plotted as geometric mean ELISA titerfor naked pCMV (solid symbols) and PLG/pCMV (open symbols) and errorbars represent SEM.

FIG. 3 is a graph depicting geometric mean neutralization titer afterDNA administration.

FIG. 4 is a graph depicting the effect of Env protein boosting on T cellresponses primed by DNA vaccines.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning. A Laboratory Manual(2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed.(Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag); Peters and Dalrymple,Fields Virology (2d ed), Fields et al. (eds.), B. N. Raven Press, NewYork, N.Y.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in theirentireties.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “an antigen”includes a mixture of two or more such antigens.

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to first set forth definitions of certain termsthat will be used hereinafter.

As used herein the term “HIV polypeptide” refers to any HIV peptide fromany HIV strain or subtype, including, but not limited to Gag, pol, env,vif, vpr, tat, rev, nef, and/or vpu; functional (e.g., immunogenic)fragments thereof, modified polypeptides thereof and combinations ofthese fragments and/or modified peptides. Furthermore, an “HIVpolypeptide” as defined herein is not limited to a polypeptide havingthe exact sequence of known HIV polypeptides. Indeed, the HIV genome isin a state of constant flux and contains several domains that exhibitrelatively high degrees of variability between isolates. As will becomeevident herein, all that is important is that the polypeptide hasimmunogenic characteristics. It is readily apparent that the termencompasses polypeptides from any of the various HIV strains andsubtypes. Furthermore, the term encompasses any such HIV proteinregardless of the method of production, including those proteinsrecombinantly and synthetically produced.

Additionally, the term “HIV polypeptide” encompasses proteins thatinclude additional modifications to the native sequence, such asadditional internal deletions, additions and substitutions (generallyconservative in nature). These modifications may be deliberate, asthrough site-directed mutagenesis, or may be accidental, such as throughnaturally occurring mutational events. All of these modifications areencompassed in the present invention so long as the modified HIVpolypeptide functions for its intended purpose. Thus, for example, in avaccine composition, the modifications must be such that immunologicalactivity is not lost. Similarly, if the polypeptides are to be used fordiagnostic purposes, such capability must be retained. Thus, the termalso includes HIV polypeptides that differ from naturally occurringpeptides, for example peptides that include one or more deletions (e.g.,variable regions deleted from Env), substitutions and/or insertions.Nonconservative changes are generally substitutions of one of the aboveamino acids with an amino acid from a different group (e.g.,substituting Asn for Glu), or substituting Cys, Met, His, or Pro for anyof the above amino acids. Substitutions involving common amino acids areconveniently performed by site specific mutagenesis of an expressionvector encoding the desired protein, and subsequent expression of thealtered form. One may also alter amino acids by synthetic orsemi-synthetic methods. For example, one may convert cysteine or serineresidues to selenocysteine by appropriate chemical treatment of theisolated protein. Alternatively, one may incorporate uncommon aminoacids in standard in vitro protein synthetic methods. Typically, thetotal number of residues changed, deleted or added to the nativesequence in the mutants will be no more than about 20, preferably nomore than about 10, and most preferably no more than about 5.

“Synthetic” polynucleotide sequences, as used herein, refers toHIV-encoding polynucleotides (e.g., Gag- and/or Env-encoding sequences)whose expression has been optimized, for example, by codon substitutionand inactivation of inhibitory sequences. See, e.g., U.S. Pat. No.6,602,705 and International Publications WO 00/39302; WO 02/04493; WO00/39303; and WO 00/39304 for examples of synthetic HIV-encodingpolynucleotides.

“Wild-type” or “native” sequences, as used herein, refers to polypeptideencoding sequences that are essentially as they are found in nature,e.g., Gag and/or Env encoding sequences as found in other isolates suchas Type C isolates (e.g., Botswana isolates AF110965, AF110967, AF110968or AF110975 or South African isolates).

As used herein, the term “virus-like particle” or “VLP” refers to anonreplicating, viral shell, derived from any of several virusesdiscussed further below. VLPs are generally composed of one or moreviral proteins, such as, but not limited to those proteins referred toas capsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art and discussed more fully below. The presence of VLPsfollowing recombinant expression of viral proteins can be detected usingconventional techniques known in the art, such as by electronmicroscopy, X-ray crystallography, and the like. See, e.g., Baker etal., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol. (1994)68:4503-4505. For example, VLPs can be isolated by density gradientcentrifugation and/or identified by characteristic density banding.Alternatively, cryoelectron microscopy can be performed on vitrifiedaqueous samples of the VLP preparation in question, and images recordedunder appropriate exposure conditions.

By “particle-forming polypeptide” derived from a particular viralprotein is meant a full-length or near full-length viral protein, aswell as a fragment thereof, or a viral protein with internal deletions,which has the ability to form VLPs under conditions that favor VLPformation. Accordingly, the polypeptide may comprise the full-lengthsequence, fragments, truncated and partial sequences, as well as analogsand precursor forms of the reference molecule. The term thereforeintends deletions, additions and substitutions to the sequence, so longas the polypeptide retains the ability to form a VLP. Thus, the termincludes natural variations of the specified polypeptide sincevariations in coat proteins often occur between viral isolates. The termalso includes deletions, additions and substitutions that do notnaturally occur in the reference protein, so long as the protein retainsthe ability to form a VLP. Preferred substitutions are those that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cystine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term “antigen” denotes both subunit antigens, (i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature), as well as, killed,attenuated or inactivated bacteria, viruses, fungi, parasites or othermicrobes. Antibodies such as anti-idiotype antibodies, or fragmentsthereof, and synthetic peptide mimotopes, which can mimic an antigen orantigenic determinant, are also captured under the definition of antigenas used herein. Similarly, an oligonucleotide or polynucleotide thatexpresses an antigen or antigenic determinant in vivo, such as in genetherapy and DNA immunization applications, is also included in thedefinition of antigen herein.

For purposes of the present invention, antigens are preferably derivedfrom any subtype of HIV. Antigens can also be derived from any ofseveral known viruses, bacteria, parasites and fungi, or tumor antigens.Furthermore, for purposes of the present invention, an “antigen” refersto a protein that includes modifications, such as deletions, additionsand substitutions (generally conservative in nature), to the nativesequence, so long as the protein maintains the ability to elicit animmunological response, as defined herein. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts that produce the antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells, Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations, or by measurementof epitope specific T-cells (e.g., by the tetramer technique) (reviewedby McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med.187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al, Immunol. Rev.150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997).

Thus, an immunological response as used herein may be one thatstimulates the production of CTLs, and/or the production or activationof helper T-cells. The HIV antigen(s) may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest. The immunogeniccomposition can be introduced directly into a recipient subject, such asby injection, inhalation, oral, intranasal and mucosal (e.g.,intra-rectally or intra-vaginally) administration.

By “subunit vaccine” is meant a vaccine composition that includes one ormore selected antigens but not all antigens, derived from or homologousto, an antigen from a pathogen of interest such as from a virus,bacterium, parasite or fungus. Such a composition is substantially freeof intact pathogen cells or pathogenic particles, or the lysate of suchcells or particles. Thus, a “subunit vaccine” can be prepared from atleast partially purified (preferably substantially purified) immunogenicpolypeptides from the pathogen, or analogs thereof. The method ofobtaining an antigen included in the subunit vaccine can thus includestandard purification techniques, recombinant production, or syntheticproduction.

“Substantially purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

A “coding sequence” or a sequence that “encodes” a selected polypeptide,is a nucleic acid molecule that is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences (or “controlelements”). The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A coding sequence can include, but is notlimited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNAsequences from viral or procaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence may be located 3′ to thecoding sequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences.

A “nucleic acid” molecule can include, but is not limited to,procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA,genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting procaryotic microorganisms or eucaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An implementation of this algorithm for nucleic acid and peptidesequences is provided by the Genetics Computer Group (Madison, Wis.) intheir BestFit utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). Other equally suitable programs for calculating thepercent identity or similarity between sequences are generally known inthe art.

For example, percent identity of a particular nucleotide sequence to areference sequence can be determined using the homology algorithm ofSmith and Waterman with a default scoring table and a gap penalty of sixnucleotide positions. Another method of establishing percent identity inthe context of the present invention is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages, theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated,the “Match” value reflects “sequence identity.” Other suitable programsfor calculating the percent identity or similarity between sequences aregenerally known in the art, such as the alignment program BLAST, whichcan also be used with default parameters. For example, BLASTN and BLASTPcan be used with the following default parameters: geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:http://www.ncbi.nlm.gov/cgi-bin/BLAST.

One of skill in the art can readily determine the proper searchparameters to use for a given sequence in the above programs. Forexample, the search parameters may vary based on the size of thesequence in question. Thus, for example, a representative embodiment ofthe present invention would include an isolated polynucleotide having Xcontiguous nucleotides, wherein (i) the X contiguous nucleotides have atleast about 50% identity to Y contiguous nucleotides derived from any ofthe sequences described herein, (ii) X equals Y, and (iii) X is greaterthan or equal to 6 nucleotides and up to 5000 nucleotides, preferablygreater than or equal to 8 nucleotides and up to 5000 nucleotides, morepreferably 10-12 nucleotides and up to 5000 nucleotides, and even morepreferably 15-20 nucleotides, up to the number of nucleotides present inthe full-length sequences described herein (e.g., see the SequenceListing and claims), including all integer values falling within theabove-described ranges.

The polynucleotides described herein include related polynucleotidesequences having about 80% to 100%, greater than 80-85%, preferablygreater than 90-92%, more preferably greater than 95%, and mostpreferably greater than 98% sequence (including all integer valuesfalling within these described ranges) identity to the sequencesdisclosed herein (for example, to the claimed sequences or othersequences of the present invention) when the sequences of the presentinvention are used as the query sequence.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al., supraor Ausubel et al., supra). Such assays can be conducted using varyingdegrees of selectivity, for example, using conditions varying from lowto high stringency. If conditions of low stringency are employed, theabsence of non-specific binding can be assessed using a secondary probethat lacks even a partial degree of sequence identity (for example, aprobe having less than about 30% sequence identity with the targetmolecule), such that, in the absence of non-specific binding events, thesecondary probe will not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” typicallyhybridizes under conditions that allow detection of a target nucleicacid sequence of at least about 10-14 nucleotides in length having atleast approximately 70% sequence identity with the sequence of theselected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al., supraor Ausubel et al., supra).

A first polynucleotide is “derived from” second polynucleotide if it hasthe same or substantially the same basepair sequence as a region of thesecond polynucleotide, its cDNA, complements thereof, or if it displayssequence identity as described above.

A first polypeptide is “derived from” a second polypeptide if it is (i)encoded by a first polynucleotide derived from a second polynucleotide,or (ii) displays sequence identity to the second polypeptides asdescribed above.

Generally, a viral polypeptide is “derived from” a particularpolypeptide of a virus (viral polypeptide) if it is (i) encoded by anopen reading frame of a polynucleotide of that virus (viralpolynucleotide), or (ii) displays sequence identity to polypeptides ofthat virus as described above.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof that is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of an antigen, antigens, an epitope, orepitopes. The nucleic acid molecule can be introduced directly into arecipient subject, such as by injection, inhalation, oral, intranasaland mucosal administration, or the like, or can be introduced ex vivo,into cells which have been removed from the host. In the latter case,the transformed cells are reintroduced into the subject where an immuneresponse can be mounted against the antigen encoded by the nucleic acidmolecule.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting DNA of interest into a host cell. Such methods canresult in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene delivery expression vectors include,but are not limited to, vectors derived from alphaviruses, pox virusesand vaccinia viruses. When used for immunization, such gene deliveryexpression vectors may be referred to as vaccines or vaccine vectors.

“T lymphocytes” or “T cells” are non-antibody producing lymphocytes thatconstitute a part of the cell-mediated arm of the immune system. T cellsarise from immature lymphocytes that migrate from the bone marrow to thethymus, where they undergo a maturation process under the direction ofthymic hormones. Here, the mature lymphocytes rapidly divide increasingto very large numbers. The maturing T cells become immunocompetent basedon their ability to recognize and bind a specific antigen. Activation ofimmunocompetent T cells is triggered when an antigen binds to thelymphocyte's surface receptors.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells. The term refers to both stableand transient uptake of the genetic material, and includes uptake ofpeptide- or antibody-linked DNAs.

Transfer of a “suicide gene” (e.g., a drug-susceptibility gene) to atarget cell renders the cell sensitive to compounds or compositions thatare relatively nontoxic to normal cells. Moolten, F. L. (1994) CancerGene Ther. 1:279-287. Examples of suicide genes are thymidine kinase ofherpes simplex virus (HSV-tk), cytochrome P450 (Manome et al. (1996)Gene Therapy 3:513-520), human deoxycytidine kinase (Manome et al.(1996) Nature Medicine 2(5):567-573) and the bacterial enzyme cytosinedeaminase (Dong et al. (1996) Human Gene Therapy 7:713-720). Cells thatexpress these genes are rendered sensitive to the effects of therelatively nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide(cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine kinase)or 5-fluorocytosine (bacterial cytosine deaminase). Culver et al. (1992)Science 256:1550-1552, Huber et al. (1994) Proc. Natl. Acad. Sci. USA91:8302-8306.

A “selectable marker” or “reporter marker” refers to a nucleotidesequence included in a gene transfer vector that has no therapeuticactivity, but rather is included to allow for simpler preparation,manufacturing, characterization or testing of the gene transfer vector.

A “specific binding agent” refers to a member of a specific binding pairof molecules wherein one of the molecules specifically binds to thesecond molecule through chemical and/or physical means. One example of aspecific binding agent is an antibody directed against a selectedantigen.

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are intended to be covered. Thesystem described above is intended for use in any of the abovevertebrate species, since the immune systems of all of these vertebratesoperate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and/or (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

“Nucleic acid expression vector” refers to an assembly that is capableof directing the expression of a sequence or gene of interest. Thenucleic acid expression vector may include a promoter that is operablylinked to the sequences or gene(s) of interest. Other control elementsmay be present as well. Nucleic acid expression vectors include, but arenot limited to, plasmids, viral vectors, alphavirus vectors (e.g.,Sindbis), eukaryotic layered vector initiation systems (see, e.g., U.S.Pat. No. 6,342,372), retroviral vectors, adenoviral vectors,adeno-associated virus vectors and the like. See, also, U.S. Pat. No.6,602,705 for a description of various nucleic acid expression vectors.Expression cassettes may be contained within a nucleic acid expressionvector. The vector may also include a bacterial origin of replication,one or more selectable markers, a signal that allows the construct toexist as single-stranded DNA (e.g., a M13 origin of replication), amultiple cloning site, and a “mammalian” origin of replication (e.g., aSV40 or adenovirus origin of replication).

“Packaging cell” refers to a cell that contains those elements necessaryfor production of infectious recombinant retrovirus that are lacking ina recombinant retroviral vector. Typically, such packaging cells containone or more expression cassettes which are capable of expressingproteins which encode Gag, pol and env proteins.

“Producer cell” or “vector producing cell” refers to a cell thatcontains all elements necessary for production of recombinant retroviralvector particles.

In addition, the following is a partial list of abbreviations usedherein:

-   -   μg microgram    -   AIDS acquired immune deficiency syndrome    -   APC antigen presenting cell    -   CCR5 chemokine receptor 5    -   CD4+ cluster of differeniation 4 receptor    -   CD8+ cluster of differeniation 8 receptor    -   CDC centers for disease control    -   CHO cells Chinese hamster ovary cells    -   CMV cytomegalovirus    -   ConA Concanvalim A    -   CRF case report form    -   CRF's circulating recombinant forms    -   CTAB cetyltrimethylamonium bromide    -   CTL cytotoxic T lymphocyte    -   Cv cromium    -   DEAE Diethylaminoethyl    -   DNA deoxyribonucleic acid    -   DTH delayed type hypersensitivity    -   ELISA enzyme-linked immunosorbent assay    -   ELISPOT enzyme-linked immunospot assay    -   ENV envelope    -   FIGE field inversion gel electrophoresis    -   GAG group-specific antigen    -   GLP good laboratory practices    -   gp glycoprotein    -   HAART highly active antiretroviral therapy    -   HAP hydroziapatic    -   HBsAg hepatitis B surface antigen    -   HCV hepatitis C virus    -   HIV/HIV-1 human immunodeficiency virus/Type 1    -   hr hour    -   HSV herpes simplex virus    -   IFN interferon    -   IFNγ interferon gamma    -   IM intramuscular    -   IND investigational new drug    -   IV intravenous    -   Kb kilobase    -   kD kilodalton    -   Kg kilogram    -   mg milligram    -   mL milliliter    -   MF59 oil-in-water emulsion adjuvant    -   NaCl sodium chloride    -   NIAID National Institute of Allergy and Infectious Disease    -   NIH National Institutes of Health    -   o- or O-oligomeric    -   PCR polymerase chain reaction    -   PEG polyethylene glycol    -   PLG cationic poly-lactide-coglycolide    -   pSIN sindbis virus vector    -   PVA poly(vinyl alcohol)    -   REV viral protein—involved in regulation of viral expression    -   SAE serious adverse event    -   SHIV simian human immunodefiency virus    -   SP resin modified polyester-carbonate resin

General Overview

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

The present invention relates to methods and compositions for thedevelopment of immunogenic compositions (e.g., vaccines) for HIV. Forexample, an HIV vaccine as described herein may include three or morecomponents. Vaccines as described herein may be intended forintramuscular injection. In certain embodiments, two nucleic acidcomponents are formulated onto (adsorbed onto) cationicpoly-lactide-coglycolide (PLG) microparticles and administered aspriming immunizations. In addition to the DNA components, a proteincomposition is also administered in one or more boosting immunizations.The protein component typically comprises at least one HIV polypeptide,for example, a CHO cell-produced, recombinant oligomeric envelopeprotein with a deletion in the V2 region mixed with the MF-59 adjuvant.

Pharmaceutic Compositions

In a preferred embodiment, the HIV vaccines described herein includesmultiple (e.g., three or more) components intended for administration(e.g., intramuscularly) in a 6-9 month, or even longer, time period. Thecomponents may be given concurrently or at different time points. Forexample, two nucleic acid “priming” immunizations may be given, whereeach priming immunization includes include two separate preparations ofDNA encoding Gag protein(s) (e.g., p55 Gag from HIV-1 SF2), and/or Envprotein(s) (e.g., an oligomeric, V2-deleted, gp140 envelope protein fromHIV-1 SF162), both formulated on PLG microparticles. The nucleic acidswill typically be provided separately in unit dose vials containingbetween 1 μg to 10 mg of DNA and between 10 μg and 100 mg of PLG (e.g.,1 mg of DNA and 25 mg of PLG microparticles). The DNA-containing dosesare typically stored in lyophilized form and vials are generallyreconstituted in the field. It should be noted that each unit dose vialwill typically contain more DNA (or protein) than is actuallyadministered to the patient. The final dosage typically consists of 1 mgin 0.5 mL each of Gag and Env DNA. The DNA components of the vaccine areintended to prime antibody, CD4 and CD8 T cell responses to HIV antigens(e.g., Gag and Env).

As noted above, the immunogenic systems (vaccines) described herein alsocomprise at least one protein component, typically an HIV polypeptidefrom any isolate or strain of HIV. For example, in certain embodiments,the protein component comprises a recombinant oligomeric envelopeprotein from the SF162 strain of HIV-1. Protein monomers of HIV Env maybe truncated to an approximate molecular size of 140 kD (e.g., toimprove solubility) and the V2 loop may be at least partially removed.The resulting oligomeric molecule resembles the envelope structure ofHIV closely. Removal of the V2 variable loop exposes conserved epitopesinvolved in receptor and/or co-receptor binding. Macaques primed withnaked DNA vaccines encoding oligomeric V2-deleted gp140 from the subtypeB (CCR5) primary isolate SF162, and boosted with the correspondingrecombinant protein, produced antibodies capable of neutralizing a rangeof distinct subtype B primary isolates. Barnett et al. (2001) J Virol.75(12):5526-40; Srivastava et al. (2002) J Virol. (6):2835-47;Srivastava et al. (2003) J. Virol. 77(20):11244-11259.

Based on the quantities of passively administered antibodies required toprotect macaques and the magnitude and breadth of the neutralizationtiters seen in macaque studies, suggest that the antibodies induced byvaccines described herein are likely to provide protection frominfection in a proportion of animals. Mascola et al. (1999) J Virol.73(5): 4009-18. The amount of protein per does can vary from microgramto milligram amounts. In certain embodiments, the protein is providedsuch that the dose administered is approximately 100 micrograms in unitdose vials containing envelope protein in sodium citrate buffer, pH 6.0without preservative.

The protein and/or nucleic acid compositions described herein may alsocomprise a pharmaceutically acceptable carrier. The carrier should notitself induce the production of antibodies harmful to the host.Pharmaceutically acceptable carriers are well known to those in the art.Suitable carriers are typically large, slowly metabolized macromoleculessuch as proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, lipid aggregates (such asoil droplets or liposomes), and inactive virus particles. Examples ofparticulate carriers include those derived from polymethyl methacrylatepolymers, as well as microparticles derived from poly(lactides) andpoly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al.,Pharm. Res. (1993) 10:362-368; McGee et al. (1997) J Microencapsul.14(2):197-210; O'Hagan et al. (1993) Vaccine 11(2):149-54. Such carriersare well known to those of ordinary skill in the art. Additionally,these carriers may function as immunostimulating agents (“adjuvants”).Furthermore, the antigen may be conjugated to a bacterial toxoid, suchas toxoid from diphtheria, tetanus, cholera, etc., as well as toxinsderived from E. coli.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art.Compositions of the invention can also contain liquids or excipients,such as water, saline, glycerol, dextrose, ethanol, or the like, singlyor in combination, as well as substances such as wetting agents,emulsifying agents, or pH buffering agents. Liposomes can also be usedas a carrier for a composition of the invention, such liposomes aredescribed above.

Briefly, with regard to viral particles, replication-defective vectors(also referred to above as particles) may be preserved either in crudeor purified forms. Preservation methods and conditions are described inU.S. Pat. No. 6,015,694.

Further, the compositions described herein can include variousexcipients, adjuvants, carriers, auxiliary substances, modulatingagents, and the like. Preferably, the compositions will include anamount of the antigen sufficient to mount an immunological response. Anappropriate effective amount can be determined by one of skill in theart. Such an amount will fall in a relatively broad range that can bedetermined through routine trials and will generally be an amount on theorder of about 0.1 μg to about 1000 μg (e.g., antigen and/or particle),more preferably about 1 μg to about 300 μg, of particle/antigen.

As noted above, one or more of the components may further comprise oneor more adjuvants. Preferred adjuvants to enhance effectiveness includeof the composition includes, but are not limited to: (1) aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) oil-in-water emulsion formulations (with or withoutother specific immunostimulating agents such as muramyl peptides (seebelow) or bacterial cell wall components), such as for example (a) MF59™(International Publication No. WO 90/14837; Chapter 10 in Vaccinedesign: the subunit and adjuvant approach, eds. Powell & Newman, PlenusPress, 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE) formulated intosubmicron particles using a microfluidizer, (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP(see below) either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) Ribi™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% Tween 80, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3)saponin adjuvants, such as QS21 or Stimulon™ (Cambridge Bioscience,Worcester, Mass.) may be used or particle generated therefrom such asISCOMs (immunostimulating complexes), which ISCOMS may be devoid ofadditional detergent (see, e.g., WO 00/07621); (4) Complete FreundsAdjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines,such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), IL16, etc.), interferons (e.g., gamma interferon), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF), betachemokines (MIP, 1-alpha, 1-beta Rantes, etc.), etc.; (6) monophosphoryllipids A (MPL) or 3-O-deacylated MPL (3dMPL) e.g., GB-222021,EP-A-0689454, optionally in the substantial absence of alum when usedwith pneumococcal saccharides e.g., WO 00/56358; (7) combinations of3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g.,EP-A-0835318, EP-A-0735898, EP-A-0761231; (8) oligonucleotidescomprising CpG motifs (Roman et al., Nat. Med., 1997, 3:849-854: Weineret al., PNAS USA, 1997, 94:10833-10837; Davis et al. J Immunol., 1998,160:870-876; Chu et al., J. Exp. Med., 1997, 186:1623-1631; Lipford etal., Eur. J. Immunol. 1997, 27:2340-2344; Moldoveanu. et al., Vaccine,1988, 16:1216-1224, Krieg et al., Nature, 1995, 3742:546-549; Klinman etal., PNAS USA, 1996, 93:2879-2883: Ballas et al., J Immunol., 1996,157:1840-1845; Cowdery et al., J Immunol., 1996, 156:4570-4575; Halpernet al., Cell. Immunol., 1996, 167:72-78; Yamamoto et al., Jpn. J. CancerRes., 1988, 79:866-873; Stacey et al., J Immunol, 1996, 157:2116-2122;Messina et al., J. Immunol., 1991, 147:17591764; Yi et al., J Immunol.,1996, 157:4918-4925; Yi et al., J Immunol., 1996, 157:5394-5402; Yi etal., J Immunol., 1998, 160:4755-4761; and Yi et al., J Immunol., 1998,1605:5898-5906; International patent applications WO96/02555,WO98/16247, WO98/18810, WO98/401005 WO98/55495, WO98/37919 andWO98/52581) i.e. containing at least one CG dinucleotide, with 5methylcytosine optionally being used in place of cytosine; (8) apolyoxyethylene ether or a polyoxyethylone ester e.g. WO 99/52549; (9) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol (WO 01/21207) or a polyoxyethylene alkyl ether or estersurfactant in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO 01/21152); (10) a saponin and animmunostimulatory oligonucleotide (e.g., a CpG oligonucleotide) (WO00/62800); (1) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and oil-in-water emulsion e.g., WO 99/11241;(13) a saponin (e.g., QS21)+3dMPL=IL-12 (optionally+a sterol) e.g., WO98/57659; (14) other substances that act as immunostimulating agents toenhance the effectiveness of the composition. Alum (especially aluminumphosphate and/or hydroxide) and MF59™ are preferred.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Administration of the pharmaceutical compositions described herein maybe by any suitable route (see, e.g., Section C). Particularly preferredis intramuscular or mucosal (e.g., rectal and/or vaginal)administration. Dosage treatment may be a single dose schedule or amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1 to 6 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by thepotency of the modality, the vaccine delivery employed, the need of thesubject and be dependent on the judgment of the practitioner.

In certain embodiments, the protein component is mixed beforeadministration with a proprietary oil-in-water emulsion adjuvant,MF59C.1 (hereafter referred to as MF59) (See, e.g., InternationalPublication No. WO 90/14837). Various subunit antigens (e.g., HCV E2,HIV gp120, HBsAg, CMV gB, and HSV 2 gD) have been combined with MF59adjuvant and administered to over 18,000 human subjects to date with anexcellent safety and tolerability profile. The protein booster isintended to amplify the primary antibody and CD4+ T cell responses inbreadth and duration and to provide a balanced response in both thehumoral and cellular compartments of the immune system, capable toachieve the prevention of HIV-1 infection.

As noted above, MF59 adjuvant has been extensively evaluated in clinicaltrials with a number of different subunit antigens, including thosederived from influenza, herpes simplex virus 2 (HSV), humanimmunodeficiency virus (HIV), cytomegalovirus (CMV), and hepatitis Bvirus (HBV) and is generally well tolerated with minimal local andsystemic adverse reactions that are transient and of mild-to-moderateseverity. Over 12,000 subjects have received influenza virus vaccinescombined with MF59 adjuvant emulsion in more than 30 clinical studies.Only two patients had serious adverse effects. Moreover, the incidenceof adverse effects depend upon the antigen used.

Prime-Boost Regimes

In certain embodiments, multiple administrations (e.g., prime-boost typeadministration) will be advantageously employed. For example, nucleicacid constructs expressing one or more HIV antigen(s) of interest areadministered. Subsequently, the same and/or different HIV antigen(s) areadministered, for example in compositions comprising the polypeptideantigen(s) and a suitable adjuvant. Alternatively, antigens areadministered prior to the DNA. Multiple polypeptide and multiple nucleicacid administrations (in any order) may also be employed.

As described herein, one exemplary prime-boost regime described hereinincludes two or more administrations of DNAs encoding one or more HIVantigens followed by one or more administrations of HIV polypeptideantigens themselves. For example, two or more administrations of HIV Gagand HIV Env DNA/PLG compositions (e.g., separate Gag and Env) may befollowed by one or more administration of HIV Env protein. HIV-1 DNAconstructs are able to stimulate the cellular and humoral arms of theimmune system and elicit immune responses capable of preventing HIV-1infection in chimpanzees. Boyer et al. (1997) Nat Med 3:526-532.Adsorption of DNA onto the surface of PLG microparticles improves DNAuptake by the antigen presenting cells (APCs), and enhance cellular andhumoral immune responses. O'Hagan et al. (2001) J Virol. 75(19):9037-43.PLG is particularly preferred to deliver DNA because the polymer isbiodegradable, biocompatible and has been used to develop several drugdelivery systems. Okada et al. (1997) Adv Drug Deliv Rev 28(1):43-70. Incertain embodiments, the ratio of DNA:PLG is between about 1 and 16 w/w% (or any value therebetween).

The “booster” component comprises an HIV protein from any HIV strain orsubtype, for example a recombinant oligomeric envelope protein from thesubtype B strain (e.g., SF2, SF162, etc.) and/or subtype C strain(Botswana strains and/or South African strains such as TV1). See, e.g.,Scriba et al. (2001) AIDS Res Hum Retroviruses 17(8):775-81; Scriba etal. (2002) AIDS Res Hum Retroviruses 18(2):149-59; Treumicht et al.(2002) J Med. Virol. 68(2):141-6. The protein monomers of the Envprotein may be truncated and the V2 loop partially removed to increasethe exposure of conserved epitopes that are more efficient to elicitcross-reactive neutralizing antibody. Without being bound by one theory,it appears that the protein booster is intended to amplify the primaryantibody and CD4+ T cell responses in breadth and duration. Barnett etal. (2001) J Virol 75(12):5526-40; Cherpelis et al. (2001) J. Virol.75(3):1547-50. The concentration of protein in each dose may vary fromapproximately 1 μg to over 1000 μg (or any value therebetween),preferably between about 10 μg and 500 μg, and even more preferablybetween about 30 μg and 300 μg.

To date, HIV vaccines as described herein have demonstrated a strongrecord of safety in preclinical studies and clinical trials. See, also,Example 4 below. No evidence of vaccine-related immunodeficiency hasbeen reported. Toxicology studies conducted in mice and rabbits with theHIV vaccine demonstrated that the vaccine was very well tolerated.Findings were consistent with studies conducted with other viral subunitvaccines or with MF59 adjuvant. Reversible local (intramuscular)inflammation is the only notable change seen with such vaccines (seeExample 4).

The goal of the HIV vaccine development program is to demonstrate thesafety and efficacy of a novel DNA-prime plus recombinant protein-boostHIV vaccine, that is capable of eliciting a combination of broad humoraland cellular responses, and preventing HIV infection or the developmentof advanced HIV disease/AIDS.

Sources of HIV Antigens

Polynucleotide sequences (e.g., for use in nucleic acid expressionconstructs) can be obtained using recombinant methods, such as byscreening cDNA and genomic libraries from cells expressing the gene, orby deriving the gene from a vector known to include the same.Furthermore, the desired gene can be isolated directly from cells andtissues containing the same, using standard techniques, such as phenolextraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al.,supra, for a description of techniques used to obtain and isolate DNA.The gene of interest can also be produced synthetically, rather thancloned. The nucleotide sequence can be designed with the appropriatecodons for the particular amino acid sequence desired. In general, onewill select preferred codons for the intended host in which the sequencewill be expressed. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature (1981) 292:756;Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem.(1984) 259:6311; Stemmer, W. P. C., (1995) Gene 164:49-53.

Next, the gene sequence encoding the desired antigen can be insertedinto a vector as described for example, in U.S. Pat. No. 6,602,705 andInternational Patent Publications WO 00/39302; WO 02/04493; WO 00/39303;and WO 00/39304, which describe suitable exemplary nucleic acidexpression vectors and methods of obtaining additional vectors useful inthe compositions and methods, described herein.

Expression constructs (e.g., plasmids) typically include controlelements operably linked to the coding sequence, which allow for theexpression of the gene in vivo in the subject species. For example,typical promoters for mammalian cell expression include the SV40 earlypromoter, a CMV promoter such as the CMV immediate early promoter, themouse mammary tumor virus LTR promoter, the adenovirus major latepromoter (Ad MLP), and the herpes simplex virus promoter, among others.Other nonviral promoters, such as a promoter derived from the murinemetallothionein gene, will also find use for mammalian expression.Typically, transcription termination and polyadenylation sequences willalso be present, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al., supra, as well as a bovine growth hormoneterminator sequence.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

Furthermore, HIV polypeptide-encoding nucleic acids can be constructedwhich include a chimeric antigen-coding gene sequences, encoding, e.g.,multiple antigens/epitopes of interest, for example derived from one ormore viral isolates. Alternatively, multi-cistronic cassettes (e.g.,bi-cistronic cassettes) can be constructed allowing expression ofmultiple antigens from a single mRNA using the EMCV IRES, or the like.

Further, the HIV antigens (and polynucleotides encoding these antigens)used in the claimed formulations may be obtained from one or moresubtypes of HIV. There are three distinct branches in the phylogenetictree of HIV-1 sequences, among these, the M (main) viruses account foralmost all of the human infections worldwide. The M-group viruses havebeen divided into 9 distinct genetic subtypes or clades (A through K).Worldwide, the subtypes A and C account for most of the infections,these subtypes are most common in southern Africa and India, The subtypeB is dominant in the American continent, Australia and Europe. Malim etal. (2001) Cell 104(4): 469-72. These subtypes are followed in frequencyby newer circulating recombinant forms (CRFs). HIV-1 displays anunprecedented genetic diversity within a subtype and even within asingle individual. Kwong et al. (2000) J Virol. 74(4): 1961-72. Thisdiversity is simply enormous when compared to the diversity found inviruses for which effective vaccines have been developed. Moore et al.(2001) J Virol. 75(13): 5721-9. Thus, though vaccines described hereinare typically developed based on dominant genetic subtypes, for HIV,effective vaccines against a specific subtype can be readily generatedusing the teachings herein.

INDUSTRIAL APPLICABILITY

The discovery that HIV was the etiological agent of AIDS in 1983-84raised hopes for the rapid development of a vaccine. More than 40candidate HIV vaccines have already been tested in phase I and IIclinical trials, and the first phase II trials are now under way in theUnited States and Thailand. Esparza, J. (2001) Bull World Health Organ.79(12): 1133-7. However, a major impediment for the development of thevaccine has been the lack of scientific evidence on the immunologicalcorrelates of protection against HIV and AIDS. Clerici et al. (1996)Immunol Lett 51(1-2):69-73. Even though most HIV infected individualsdevelop broad immunological responses against the virus, these responsesare incapable of eliminating the infection or preventing diseaseprogression. This problem is further complicated by the fact that HIVstrains vary significantly in different parts of the world. HIV exhibitsextensive genetic sequence heterogeneity, particularly in the genesencoding for viral envelope proteins. Different subtype viruses cancombine among themselves, generating additional circulating recombinantforms (CRFs). McCutchan et al. (1996) J. Virol. 70(6):3331-3338.

Using vaccination to induce a specific anti HIV-1 immune response thatis more effective than the natural response to the HIV-1 infection hasproven difficult to achieve. In most of the infections for whichvaccines are effective, viremia or bacteremia is a critical phase thatpermits the immune system to contain the pathogen before it reaches thetarget organ. Ada et al. (2001) New Engl. J. Med. 345:1042-1053. Thus,it has been postulated that the lack of adequate immune control of HIV-1is likely due to several factors, including HIV-1's ability to infectand deplete CD4+ cells, the main target during the initial phase ofviremia (Greene et al. (2002) Nat. Med. 8(7):673-80); HIV-1's ability tomutate the sequence of its surface antigens rapidly; the fact that HIV-1is a weak immunogen that has the ability to mask surface epitopes thatwould otherwise be recognized by neutralizing antibodies; and/or thefact that HIV can evade cellular immune responses and establish latentinfection at sites that are inaccessible to the immune system (Gotch etal. (2000) Curr Opin Infect Dis 13(1):13-17).

Further, although most licensed vaccines elicit both cellular andantibody responses, little is understood about how these known vaccinesactually protect against infection. It has been postulated thatfunctional antibody responses can eliminate the inoculum either bykilling bacteria, inactivating viruses or neutralizing toxins. Plotkinet al. (2001) Pediatr Infect Dis J. 20(1):63-75. However, previously,the HIV vaccines tested have not been able to elicit adequate titers ofHIV-1 specific broad neutralizing antibodies against diverse primaryisolates of HIV-1.

Among the scientific community, there is general agreement that in orderto be successful, an HIV/AIDS vaccine should i) induce antibodies ableto neutralize a broad range of primary isolates, ii) induce a durableCD8+ mediated cytotoxic response against a variety of strains, and iii)induce a strong CD4+ T cell response to sustain the CTL activity. See,e.g., Mascola et al. (1999) J Virol 73(5): 4009-18. Passivelyadministered antibodies alone can protect macaques against both mucosaland IV challenges with pathogenic SHIV. See, e.g., Mascola et al. (2001)Curr Opin Immunol 13(4):489-95. There is, however, skepticism thatbroadly cross-reactive neutralizing antibodies can be elicited in humansby immunization. This has led some investigators to abandon efforts toinclude envelope in their vaccines and promote vaccines that relyexclusively on cellular immunity for protection. See, also, Kaul et al.(2001) J Clin Invest 107(3): 341-9). However, such vaccines are unlikelyto protect from infection and may be expected to limit diseaseprogression.

Thus, the compositions and methods described herein preferably elicit acombination of humoral (neutralizing antibody) and cellular (CD4+ Tcells and CD8+ T Cells) responses, although humoral or cellularresponses individually may be sufficient. The priming regimen ispreferably based on nucleic acid vectors (e.g., pCMV or pSIN) thatcomprise Gag and/or Env HIV genes, respectively. DNA-based vaccines areattractive because they are flexible and relatively simple to produce.Their distribution may be simplified because DNA itself is very durablewhen properly stored. Immunization with DNA encoding antigenic proteinselicits both antibody and cell-mediated immune responses. DNAimmunization has provided protective immunity in various animal models.See, e.g., Donnelly et al. (1997) Life Sci. 60(3):163-72, A DNA vaccineencoding a malaria antigen was tolerated relatively well by 20volunteers, with only few and mild local reactogenicity and systemicsymptoms. Wang et al. (1998) Science 282(5388):476-80. A DNA-basedvaccine containing HIV-1 Env and Rev genes was administered to 15asymptomatic HIV-infected patients who were not using antiviral drugs.The vaccine induced no local or systemic reactions, and no laboratoryabnormalities were detected. Specifically, no patient developedautoimmune antibodies. MacGregor et al. (1998) J Infect Dis178(1):92-100. Ongoing Phase 1 clinical trials show that therapeuticvaccinations indeed boost anti-HIV-1 immune responses in humans. Ugen etal. (1998) Vaccine 16(19):1818-21.

The boost component of the compositions and methods described hereintypically includes an HIV protein (e.g., a HIV envelope gp140 proteinthat has a deletion of the V2 loop, thus exposing conserved epitopes).The HIV protein vaccines described herein generally comprise subunitrecombinant antigens and are predicted to be both well tolerated andimmunogenic (humoral and cellular) in view of the safety and efficacydate obtained with non-recombinant HIV protein vaccines.

Formulations and Administration

As noted above, the compositions are preferably administered using a“prime-boost” approach, for example, two priming injections (e.g., eachincluding two separate preparations of DNA encoding p55 Gag from HIV-1SF2, and oligomeric, V2 loop-deleted, gp140 envelope protein from HIV-1SF162, both formulated on PLG microparticles (Env or Gag PLG/DNA)) areadministered. The boost composition comprises a protein, for example anantigen is composed of a recombinant oligomeric, V2 loop-deleted, gp140envelope protein (HIV o-gp140) in combination with MF59 adjuvant. Theprotein is typically mixed with the adjuvant shortly before injection.

The DNA vaccines may be provided in 5.0 mL Type I glass vials containing1.4 mg of DNA and 35 mg of PLG microparticles per vial, in lyophilizedform. HIV o-gp140 antigen is supplied as a liquid in 3-mL Type I glassvials containing 140 μg in 0.35 mL/vial. MF59 adjuvant is supplied in3-mL Type I glass vials containing 0.7 mL/vial. Generally, the dose ofDNA and protein actually administered to the subject is less thancontained in the vial, for example approximately 1.0 mg of DNA istypically administered to the subject when the vial contains 1.4 mg.Similarly, approximately 100 μg of protein is typically administered tothe subject when each unit dose vial contains 140 μg of protein.

Any suitable delivery mode can be used for the nucleic acids andpolypeptides. Liposomes can also be used for delivery of thesemolecules. For a review of the use of liposomes as carriers for deliveryof nucleic acids, see, Hug and Sleight, Biochim. Biopllys. Acta. (1991)1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol.101, pp. 512-527. Liposomal preparations for use in the presentinvention include cationic (positively charged), anionic (negativelycharged) and neutral preparations, with cationic liposomes particularlypreferred. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA(1989) 86:6077-6081); and purified transcription factors (Debs et al.,J. Biol. Chem. (1990) 265:10189-10192), in functional form. Cationicliposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include (DDAB/DOPE)and DOTAP/DOPE (Boerhinger).

Similarly, anionic and neutral liposomes are readily available, such as,from Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The DNA and/or protein antigen(s) can also be delivered in cochleatelipid compositions similar to those described by Papahadjopoulos et al.,Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos.4,663,161 and 4,871,488.

The vaccine components may also be encapsulated, adsorbed to, orassociated with, particulate carriers. Such carriers present multiplecopies of a selected antigen to the immune system and promote trappingand retention of antigens in local lymph nodes. The particles can bephagocytosed by macrophages and can enhance antigen presentation throughcytokine release. Examples of particulate carriers include those derivedfrom polymethyl methacrylate polymers, as well as microparticles derivedfrom poly(lactides) and poly(lactide-co-glycolides), known as PLG. See,e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al.,J Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine11(2): 149-54, 1993. Suitable microparticles may also be manufactured inthe presence of charged detergents, such as anionic or cationicdetergents, to yield microparticles with a surface having a net negativeor a net positive charge. For example, microparticles manufactured withanionic detergents, such as hexadecyltrimethylammonium bromide (CTAB),i.e. CTAB-PLG microparticles, adsorb negatively charged macromolecules,such as DNA. (see, e.g., Int'l Application Number PCT/US99/17308).Methods of making and using PLG particles to deliver nucleic acids aredescribed in International Patent Publications WO 98/33487; WO 00/06123;WO 02/26212; and WO 02/26209.

Polymers such as polylysine, polyarginine, polyornithine, spermine,spermidine, as well as conjugates of these molecules, may also be usedfor transferring a nucleic acid of interest.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for delivering nucleicacid vectors of the present invention. The particles are coated with thenucleic acid(s) to be delivered and accelerated to high velocity,generally under a reduced atmosphere, using a gun powder discharge froma “gene gun.” For a description of such techniques, and apparatusesuseful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006;5,100,792; 5,179,022; 5,371,015; and 5,478,744. Also, needle-lessinjection systems can be used (Davis, H. L., et al, Vaccine12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

The compositions described herein may either be prophylactic (to preventinfection) or therapeutic (to treat disease after infection). Thecompositions will comprise a “therapeutically effective amount” of thegene of interest such that an amount of the antigen can be produced invivo so that an immune response is generated in the individual to whichit is administered. The exact amount necessary will vary depending onthe subject being treated; the age and general condition of the subjectto be treated; the capacity of the subject's immune system to synthesizeantibodies; the degree of protection desired; the severity of thecondition being treated; the particular antigen selected and its mode ofadministration, among other factors. An appropriate effective amount canbe readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials.

The compositions will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present in such vehicles.Certain facilitators of nucleic acid uptake and/or expression can alsobe included in the compositions or coadministered, such as, but notlimited to, bupivacaine, cardiotoxin and sucrose.

Once formulated, the compositions of the invention can be administereddirectly to the subject (e.g., as described above) or, alternatively,delivered ex vivo, to cells derived from the subject, using methods suchas those described above. For example, methods for the ex vivo deliveryand reimplantation of transformed cells into a subject are known in theart and can include, e.g., dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, lipofectamineand LT-1 mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) (with or without thecorresponding antigen) in liposomes, and direct microinjection of theDNA into nuclei.

Direct delivery of polynucleoides and polypeptides in vivo willgenerally be accomplished, as described herein, by injection usingeither a conventional syringe or a gene gun, such as the Accell® genedelivery system (PowderJect Technologies, Inc., Oxford, England). Theconstructs can be injected either subcutaneously, epidermally,intradermally, intramucosally such as nasally, rectally and vaginally,intraperitoneally, intravenously, orally or, preferably,intramuscularly. Dosage treatment may be a single dose schedule or amultiple dose schedule. Administration of nucleic acids may also becombined with administration of peptides or other substances.

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXPERIMENTAL Example 1 Vaccine Manufacturing Process and Release

A. PLG/DNA HIV Vaccines

For PLG/DNA priming immunization with nucleic acid, plasmid DNA (Env orGag) was adsorbed onto biodegradable polymer microparticles (PLG)essentially as follows. To manufacture the DNA vaccines, E. coli (strainDH5) was transformed with plasmids encoding the HIV Env and Gag genes. Amodified alkaline lysis method was used to isolate plasmid DNA fromchromosomal DNA, proteins, and other cellular debris. Plasmid DNA wasconcentrated by precipitation using PEG 8000. The plasmids were thenpurified by two chromatography steps and transferred by ultrafiltrationinto formulation buffer.

PLG microparticles were produced by an aseptic manufacturing process.See, e.g., U.S. Pat. Nos. 5,603,960; 6,534,064 and 6,573,238; Gupta etal. (1998) Adv Drug Deliv Rev. 32(3):225-246; O'Hagan (1998) J PharmPharmacol. 50(1): 1-10. In particular, PLG (dissolved in methylenechloride) was homogenized with formulation buffer and CTAB (cationsurfactant) solution under high speed and high shear of mixing to form astable emulsion. The removal of methylene chloride by nitrogen purgecauses PLG to form microparticles, due to the tendency of the cationicsurfactant to stay at the PLG interface. These positively chargedmicroparticles bind with negatively charged DNA to form the PLG/DNAimmunogen.

B. HIV o-gp140 Antigen

The recombinant, oligomeric HIV gp-140 (o-gp140) was preparedessentially as described in Srivastava et al. (2003) J Virol.77(20):11244-11259. Following fermentation of the host cells, the cellculture supernatant was harvested, filtered, concentrated, and purified.

The purified o-gp140 protein fraction was further treated to removeadventitious viruses. The first of these steps was viral inactivation atpH 3.5 for 1 hour. The sample was then concentrated and diafiltered intoa buffer at pH 4 in preparation for cation capture using SP resin, whichcaptures o-gp140 and allows many viruses to flow through. The o-gp140was eluted, concentrated and diafiltered into formulation buffer. Thisformulated bulk product was then filtered through a Ultipor® VF gradeDV50 virus removal membrane followed by filtration through a 0.2 μmmembrane.

C. MF59 Adjuvant

MF59 adjuvant (MF59C.1) is an oil-in-water emulsion with a squaleneinternal oil phase and a citrate buffer external aqueous phase. See,e.g., U.S. Pat. Nos. 6,299,884 and 6,086,901; Ott et al. “MF59—Designand Evaluation of a Safe and Potent Adjuvant for Human Vaccines,”Vaccine Design: The Subunit and Adjuvant Approach (Powell, M. F. andNewman, M. J. eds.) Plenum Press, New York, pp. 277-296 (1995). Twononionic surfactants, sorbitan trioleate and polysorbate 80, serve tostabililize the emulsion. The safety of the MF59 adjuvant has beendemonstrated in animals and in humans in combination with a number ofantigens. See, e.g., Higgins et al., “MF59 Adjuvant Enhances theImmunogenicity of Influenza Vaccine in Both Young and Old Mice,” Vaccine14(6):478-484 (1996).

Example 2 Vaccine Composition

The components of the PLG/DNA priming vaccines, o-gp140 boost antigen,and MF59 adjuvant are provided in the following tables.

TABLE 1 PLG DNA (Env) Vaccine Composition Quantity Quantity per doseComponent per mL* (maximum dose) Poly (D.L-Lactide-co-glycolide) 50.0 mg25.0 mg Plasmid DNA (Env) 2.0 mg 1.0 mg HexadecyltrimethylammoniumBromide 0.5 mg 0.25 mg Mannitol, USP, EP 44 mg 22 mg Sucrose, USP/NF14.7 mg 7.35 mg EDTA, Disodium salt Dihydrate, USP 0.37 mg 0.28 mgSodium Citrate Dihydrate, USP/EP 1.4 mg 1.10 mg Citric Acid Monohydrate,USP/EP 0.04 mg 0.02 mg Water for Injection qs qs *followingreconstitution

TABLE 2 PLG DNA (Gag) Vaccine Composition Quantity Quantity per doseComponent per mL* (maximum dose) Poly (D.L-Lactide-co-glycolide) 50.0 mg25.0 mg Plasmid DNA (Gag) 2.0 mg 1.0 mg HexadecyltrimethylammoniumBromide 0.5 mg 0.25 mg Mannitol, USP, EP 44 mg 22 mg Sucrose, USP/NF14.7 mg 7.35 mg EDTA, Disodium salt Dihydrate, USP 0.37 mg 0.18 mgSodium Citrate Dihydrate, USP/EP 1.4 mg 0.70 mg Citric Acid Monohydrate,USP/EP 0.04 mg 0.02 mg Water for Injection qs qs *followingreconstitution

TABLE 3 HIV o-gp140 Antigen Composition Quantity Quantity per doseComponent per mL (100 μg) o-gp140 0.4 mg 0.1 mg Sodium citrate,dihydrate 2.75 mg 0.69 mg Citric acid, monohydrate 0.15 mg 0.04 mgSodium chloride 17.53 mg 4.38 mg Water for Injection qs qs

TABLE 4 MF59C.1 Adjuvant Composition Quantity Component per mL Quantityper dose Squalene 39 mg 9.75 mg Polysorbate 80 4.7 mg 1.18 mg Sorbitantrioleate 4.7 mg 1.18 mg Sodium citrate, dihydrate, USP 2.68 mg 0.66 mgCitric acid, monohydrate, USP 0.17 mg 0.04 mg Water for Injection qs qs

The schedule for vaccination injections is to inject at multiple timepoints (e.g., at 5 or 6 different time points), administered at 0, 1, 2,6, 9 and possibly 12 months. Several immunization schedules areevaluated to maximize the immune response. These schedules may includevaccinations at 4 or 5 timepoints, according to any schedule, forexample as set forth below. All vaccinations will be administered byintramuscular injection in the outpatient setting. Table 5 shows anexemplary immunization protocol.

TABLE 5 Protocol Of Immunization Month (day) DNA Dose Protein 0 1 2 4 69 # Gag/Env (μg) Dose (0) (28) (56) (112) (168) (236) 12* (365) 11000/1000 100 μg A A A B B B 2 1000/1000 100 μg A A A + B B B 31000/1000 100 μg A A A + B B B 4 1000/1000 100 μg A A B B B # schedule*If needed to sustain an immunologic response STUDY AGENTS A: Clade BGag + Env DNA/PLG microparticles, dose indicated below B: Clade B gp140Env protein, 100 μg

Example 3 Handling and Storage

To prepare the DNA/PLG vaccine for administration, one vial of eachDNA/PLG (Env or Gag) is reconstituted by drawing 0.7 mL Water forInjection into a syringe and adding it to each of the two vials. Thevials are swirled vigorously for up to two minutes. The mixing iscomplete when the suspensions appear uniform, milky, and fullydispersed. The reconstituted solutions are administered without furtherpreparation to deliver the highest DNA/PLG dose (1000 μg). To preparethe 500-μg, and 250-μg doses, use a new syringe to add an additional 0.7mL or 2.1 mL of 0.9% NaCl solution (Normal Saline), respectively, to theall ready reconstituted vials, and swirl to mix. Using a new syringe,draw up 0.5 mL of the Env PLG/DNA mixture, and then 0.5 mL of the GagDNA/PLG mixture, into the same syringe. The total DNA dose, in acombined volume of 1 mL, can then be administered intramuscularly (IM)into the deltoid muscle.

HIV o-gp140 antigen will be mixed before administration with MF59adjuvant. To prepare the vaccine dose for administration, mix thecontents of the MF59 vial by repeated gentle swirling and inversion (notvigorous shaking) and then withdraw 0.35 mL into a 1-mL sterile syringe.Inject this adjuvant into the 3 mL vial containing the thawed HIV o-gp140 antigen and mix by gentle swirling. Use a new syringe to draw up 0.5mL of the mixture, which can then be administered intramuscularly (IM)into the deltoid. The final vaccine has a milky white opacity. Theinjection should be given shortly after addition of the adjuvant.

The thawed HIV o-gp140 antigen is stable at 2° to 8° C. for 8 hours.Antigen that has been thawed for over 8 hours (even with refrigeration),is not preferred, as it may have reduced potency.

Individuals receiving placebo will receive 0.5 mL of calcium- andmagnesium-free phosphate-buffered saline. Supplied as a clear, colorlesssolution in vials containing a volume to deliver a 1 mL dose. The vialsmust be stored in a refrigerator at 2 to 8° C.

A. Vaccine Storage Conditions

The lyophilized DNA/PLG vaccines are stored at 2-8° C. HIV o-gp140 arestored frozen below 60° C. and the MF59 adjuvant is stored in arefrigerator at 2° to 8° C. MF59 should not be frozen.

Example 4 Animal Studies

A nonclinical safety assessment program was designed to support theclinical administration of three intramuscular (IM) doses of the HIV DNAvaccine formulation followed by three IM doses of the HIV Proteinvaccine formulation. One clinical dose (1.0 mL) of the DNA vaccineformulation contains 1 mg Env-DNA, 1 mg Gag-DNA, and 50 mg PLG whereasone clinical dose (0.5 mL) of the HIV Protein Vaccine contains 0.1mg/0.25 mL Env protein plus 0.25 mL MF59.

The following GLP studies were conducted to assess whether integrationinto host genomic DNA occurs and to characterize tissue localization andpersistence of the HIV DNA vaccine formulation when administered as asingle IM injection to New Zealand White rabbits and BALB/c mice,respectively. These studies are further described below in Section Atitled “An Integration Study with DNA-PLG Formulations after a SingleIntramuscular Injection to New Zealand White Rabbits” and Section Btitled “Single Dose Biodistribution Study of HIV DNA VaccineFormulations in BALB/c Mice.”

As described in further detail below, in these studies, toxicity wasevaluated based on viability, clinical observations, body weights, andmacroscopic postmortem examinations. Physical examinations and dermalscoring of injection sites were also performed in the mousebiodistribution study. Results of these studies demonstrated thatadministration of a single dose of the Env-DNA vaccine formulationresulted in no integration into the rabbit genomic DNA and goodtolerability in New Zealand White rabbits and BALB/c mice. The analysisof mouse tissues for distribution of the HIV DNA vaccine formulation wasalso performed.

In addition, the following GLP toxicology study was conducted to assessthe systemic and local tolerability of the HIV vaccine formulation whenadministered to New Zealand White rabbits via IM injection. (See,Section C below, titled “Multiple-Dose Intramuscular Injection ToxicityStudy with HIV DNA Vaccine Formulation in New Zealand White Rabbits”).In this study, animals received four doses, two weeks apart, of the HIVDNA vaccine formulation followed by four doses, two weeks apart, of theHIV Protein vaccine formulation. The first HIV Protein vaccine dose wasadministered on the same day as the last HIV DNA vaccine dose. Arecovery period of two weeks was included in the study design. Rabbitsreceived the planned clinical dose (1 mL HIV DNA vaccine/dose; 0.5 mLHIV Protein vaccine/dose) by the clinical route of administration (IM).However, rabbits received four doses each of the HIV DNA vaccine and theHIV Protein vaccine, exceeding the intended clinical regimen (threedoses each) by one dose. The rabbit dosing regimen was condensedrelative to the clinical regimen (monthly), however, rabbitimmunogenicity studies have demonstrated that an every two-week regimenis appropriate from an immunological standpoint.

In this study, toxicity was evaluated based on clinical signs, dermalscoring of injection sites, body weights and temperatures, foodconsumption, opthalmoscopy, clinical pathology (hematology, serumchemistry, and coagulation including fibrinogen), organ weights, andmacroscopic postmortem and histopathological examinations. Analysis ofserum for antibodies (anti-nuclear and Env- and Gag-antibodies) was alsoperformed. Under the conditions of this study and based on the availablepreliminary data (terminal organ weights, macroscopic evaluation andhistology pending), no systemic or local effects related to theadministration of the HIV vaccine formulation were identified.

The safety and persistence at the injection site of the HIV DNA vaccineformulation was further assessed in the following non-GLP studies,described in further detail below in Section D titled “ExploratoryDNA/PLG Local Irritation Tolerance Study in Male New Zealand WhiteRabbits” and Section E titled “Single Dose Intramuscular andMultiple-Dose (Two) Mouse Immunogenicity Study with PCR Injection SiteAssessment.”

The single dose study was conducted to evaluate the potential localirritant effects of various concentrations of DNA/PLG in New ZealandWhite male rabbits when administered by a single IM injection. Potentialtoxicity was evaluated based on clinical signs, dermal scoring ofinjection sites, body weight, comprehensive macroscopic examination, andmicroscopic evaluation of injection sites. Under the conditions of thisstudy, various concentrations of DNA/PLG were well tolerated whenadministered to male New Zealand White rabbits as a single IM injection.

The multiple-dose immunogenicity study assessed the presence of Gag-DNAPLG at the IM injection site four and eight weeks post-last dose infemale BALB/c mice that received two administrations (Days 0 and 28) ofGag-DNA PLG formulations. Results demonstrated that the PLG formulationswere comparable to a naked-DNA control with regard to persistence andthat the amount remaining in the injection site 4 and 8 weeks post-lastdose was insignificant (approximately 10⁻⁷% of the infected amount).

A. An Integration Study with DNA-PLG Formulations after a SingleIntramuscular Injection to New Zealand White Rabbits

To assess the integration of the HIV DNA-PLG vaccine formulation(Env-DNA PLG and Gag-DNA PLG) into the host genomic DNA whenadministered via a single IM injection to New Zealand White rabbits thefollowing studies were performed. The study consisted of three groups of2 animals/sex/group. On Day 0, treated rabbits received a single IMinjection (0.5 mL/leg) of either the Env-DNA PLG or the Gag-DNA PLG ineach hind leg. (See, Table 6). Control rabbits received no injection.All animals were necropsied on Day 29.

TABLE 6 Experimental Study Design Treatment Number of Animals Dose^(a)Volume^(b) Total Necropsy^(c) Group Material DNA (mg) (mL) M F M F 1Control 0 0 2 2 2 2 2 Env-DNA 2 1 2 2 2 2 PLG 3 Gag-DNA 2 1 2 2 2 2 PLG^(a)Group 2 and 3 animals received a dose of 1 mg DNA, 25 mg PLG/0.5mL/leg in each hind leg for a total dose/animal of 2 mg DNA, 50 mg PLG.Dosing occurred on Day 0 of the study. ^(b)Group 2 and 3 animalsreceived a volume of 0.5 mL/leg for a total volume/animal of 1 mL.^(c)Necropsy was performed 30 days post-dosing (Day 29)

Potential toxicity was evaluated based on viability observations formortality and general condition, body weights, and a comprehensivepostmortem macroscopic examination. In addition, injection sites werecollected at necropsy for Polymerase Chain Reaction (PCR) analysis toevaluate the integration of the DNA vaccine into the rabbit genomic DNA.Additional tissues (see Table 7) were also collected for potential PCRanalysis in the event of positive integration results at the injectionsite. For the PCR analysis, DNA was extracted from the rabbit tissue,quantitated, and subjected to field inversion gel electrophoresis (FIGE)to separate the rabbit genomic DNA from the extrachromosomal plasmidDNA. DNA of a size greater than 17 kb was excised and purified from thegel. Both the extracted and the FIGE purified DNAs (1 μg) were analyzedusing a quantitative PCR assay to assess the integration of the targetsequence (plasmid vector Env-DNA PLG) in each preparation. DNA extractedfrom tissues of control animals was pooled according to sex; DNA fromtreated animals was not pooled but analyzed separately.

TABLE 7 Tissues collected for PCR analysis Bone marrow (sternum, femur)Lungs (with mainstem bronchi) Brain (medulla, pons, cerebrum, Lymphnodes (submandibular) cerebellum) Kidneys Ovaries Injection Sites SpleenLiver Testes

There were no deaths and no treatment-related adverse effects onclinical signs and body weights. No treatment related changes were notedin the macroscopic examination either. Results of the PCR integrationanalysis revealed no integration of the Env-DNA PLG into the hostgenomic DNA (see Table 8). Because no integration occurred at theinjection sites, no additional tissues were evaluated.

TABLE 8 Quantitative PCR assay results of injection sites Env-DNA PLG(copies/μg DNA) SAMPLE Extracted DNA^(a) FIGE Purified DNA^(b) ControlMale LLD LLD Male # 2020 2637 LLD Male # 2021 2364 LLD Control FemaleLLD LLD Female # 2520 33890 LLD Female # 2521 19814 LLD^(q)uantification of the target sequence in genomic DNA prior to fieldinversion gel electrophoresis (extrachromosomal plasmid DNA plus genomicDNA) ^(q)uantification of the target sequence in genomic DNA purified byfield inversion gel electrophoresis (genomic DNA only) LLD = lower thatthe limit of detection of the assay (5 copies/μg DNA)

In conclusion, a single IM dose of either Env-DNA PLG or Gag-DNA PLGcontaining a total of 2 mg of DNA and 50 mg of PLG were well toleratedin New Zealand White rabbits. No treatment-related adverse effects werenoted and no integration of plasmid vector Env-DNA into rabbit genomicDNA obtained from the injection sites was detected.

B. Single Dose Biodistribution Study of HIV DNA Vaccine Formulations inBALB/c Mice

To assess the tissue localization and persistence of the HIV DNA PLGvaccine formulations (Env-DNA PLG and Gag-DNA PLG) after a singleadministration via IM injection to BALB/c mice, the following studieswere performed. The study included five groups of 15 animals/sex/group.On Day 1, treated mice received a single IM injection of either a highor a low dose of Env-DNA PLG or Gag-DNA PLG in the right biceps femorisarea. Control mice received no injection. Five animals/sex/group werenecropsied one week (Day 8), two months (Day 61), or three months (Day91) post-dosing. (Table 9).

Potential toxicity was evaluated based on viability observations formortality and general condition, physical examinations, body weights,dermal Drazie scoring of injection sites, and a comprehensive postmortemmacroscopic examination. In addition, selected tissues (see Table 10)were collected at each necropsy for PCR analysis to evaluate thebiodistribution and persistence of the DNA vaccine into mouse tissues.For the PCR analysis, DNA was extracted from each mouse tissue,quantitated, subjected to PCR amplification using a fluorescence probe,and followed by fluorescence detection. Of the collected tissues, onlytissues from the Env-DNA PLG treated rabbits were analyzed.

TABLE 9 Experimental Study Design Dose volume Number of Animals/SexGroup and Dose (μL/ Day 8 Day 61 Day 91 Treatment Level^(a) dose)^(a)Total Necropsy Necropsy Necropsy 1 0 0 15 5 5 5 (Control) None 2 10 μg20 15 5 5 5 Env-DNA DNA PLG 0.25 mg PLG 3 100 μg 50 15 5 5 5 Env-DNA DNAPLG 2.5 mg PLG 4 10 μg 20 15 5 5 5 Gag-DNA DNA PLG 0.25 mg PLG 5 100 μg50 15 5 5 5 Gag-DNA DNA PLG 2.5 mg PLG ^(a)Dosing occurred on Day 1 ofthe study.

TABLE 10 Tissues Collected for PCR Analysis Bone marrow (both femurs)Lung Brain Lymph nodes (mandibular) Kidneys Ovaries Injection Site(right biceps femoris) Spleen Liver Testes

There were no deaths that could be associated with administration of thetest articles and no treatment-related adverse effects on clinical signsand body weights. No erythema or edema was seen at the injection sites.No treatment related changes were noted in the macroscopic examination.

In conclusion, a single IM dose of either Env-DNA PLG or Gag-DNA PLGcontaining up to 100 μg of DNA and up to 2.5 mg of PLG was welltolerated in BALB/c mice. No treatment-related adverse effects werenoted.

C. Multiple-Dose Intramuscular Injection Toxicity Study with HIV DNAVaccine Formulation in New Zealand White Rabbits

To assess the local and systemic toxicity of the HIV Vaccine formulationin New Zealand White rabbits after repeated administration and todetermine the reversibility of findings, the following studies wereconducted. Two groups of 8 animals/sex/group were used. Treated rabbitsreceived four doses of the HIV DNA vaccine formulation (Env- and Gag-DNAPLG) given every other week followed by four doses of the HIV ProteinVaccine formulation, also given every other week. The last HIV DNAvaccine dose and the first HIV Protein vaccine dose were administered onthe same day (Day 43). Doses were administered via IM injections intothe quadricep leg muscle and legs were alternated except on Day 43 whenboth legs were injected. Control animals received four IM injections ofsaline solution followed by four IM injections of MF59. Fouranimals/sex/group were necropsied three days (Day 88, main necropsy) ortwo weeks post-dosing (Day 99, recovery necropsy). Table 11 describesthe experimental design.

Potential toxicity was evaluated based on clinical signs, dermal scoringof injection sites, body temperature, body weight, food consumption,ophthalmic examination, clinical pathology (hematology, coagulation, andserum chemistry parameters), terminal organ weights, comprehensivemacroscopic examination, and microscopic evaluation of selected tissues.

TABLE 11 Experimental Study Design DAY OF STUDY Treatment 1 15 29 43 5771 85 88 99 GROUP 1^(a) Control (dose volume) Saline 1 mL 1 mL 1 mL   1mL none none none Main Recovery Control Necropsy^(e) Necropsy^(e) MF59none none none 0.5 mL 0.5 mL 0.5 mL 0.5 mL Control^(b) GROUP 2^(a) DNAVaccine (dose volume) Env- & 1^(st) 2^(nd) 3^(rd) 4^(th) none none noneGag-DNA dose dose dose dose PLG^(c) (1 mL) (1 mL) (1 mL)   (1 mL) EnvProtein none none none 1^(st) 2^(nd) 3^(rd) 4^(th) Dose^(d) dose dosedose dose (0.5 mL) (0.5 mL) (0.5 mL) (0.5 mL) ^(a)16 animals (8 M + 8 F)^(c)consists of 0.25 mL of MF59 plus 0.25 mL saline ^(c)consists of 0.5mL Env-DNA PLG (2 mg DNA, 50 mg PLG/mL) plus 0.5 mL Gag-DNA PLG (2 mgDNA, 50 mg PLG/mL) ^(d)consists of 0.25 mL of Env Protein (0.4 mg/mL)plus 0.25 mL MF59 ^(e)4 animals/sex/group

The animals were observed twice daily for mortality and morbidity andonce daily for signs of toxicity. In addition, detailed observationswere made predose, 4 hr post-dose on each dosing day, weekly, and ateach necropsy. Injection sites were assessed for signs of irritation andgraded based on a modified Draize score prior to dosing and 24 and 48 hrafter each injection. Body temperatures were taken pre-treatment, priorto each dose, and 24 hr after each dose. Body weights were recordedpre-treatment, weekly thereafter, and at necropsy. Food consumption wasassessed weekly. The opthalmology evaluation was performed pre-treatmentand prior to each necropsy. Blood samples for hematology, serumchemistry, and coagulation (including fibrinogen) analysis werecollected pre-treatment, pre-dose on Days 29 and 57, and on Days 87 and99. Additional blood samples were taken pre-treatment, pre-dose on Days15, 43, 71, and on Days 87 and 99 for antibody (anti-nuclear and Env-and Gag-antibodies) analysis. At each necropsy, a complete macroscopicexamination and microscopic evaluation of selected tissues (see Table12) were performed. Organ weight data on selected organs (Table 13) werealso collected. In addition, selected tissues were collected forpossible assessment of distribution of the DNA vaccine into host tissuesby PCR analysis (Table 14).

TABLE 12 Histopathology Tissue List Eyes Kidneys Femur with bone marrowLiver (including knee joint) Gall Bladder Lung and bronchi Lesions (ifany) Optic nerve Lymph nodes (inguinal, lumbar, Spleen mesenteric, andpopliteal) Injection Sites Thymus

TABLE 13 Organ Weights List Adrenals Heart Spleen Brain Kidneys TestisEpididymis Liver Thymus Gall Bladder Ovaries

TABLE 14 Tissues Collected for Potential PCR Analysis Brain Spleen LungMandibular lymph node Liver Injection Sites Ovaries/Testis Kidney Bonemarrow

Preliminary data (up to Day 84) revealed no deaths that could beassociated with administration of the test articles and notreatment-related adverse effects on clinical signs, body weights, foodconsumption, and body temperature. Dermal scoring of the injection sitesrevealed occasional instances of edema or erythema in a few animals fromboth the control and treated group. Although the incidence of thesedermal irritation reactions was slightly higher in Group 2 (HIV Vaccinetreatment) animals, the findings were mild in severity (very slight toslight) and completely resolved by the next observation period.Available preliminary data (up to Day 57) for clinical pathologydemonstrated that there were no treatment-related effects on hematology,coagulation, or clinical chemistry parameters.

In conclusion, under the conditions of this study and based on theavailable preliminary data, no systemic effects related to theadministration of the HIV vaccine formulation were identified. Localeffects consisted of occasional instances of very slight to slighterythema or edema at the injection sites, which appeared fully resolvedby the next observation period. Four IM injections of the HIV DNAvaccine given every other week, followed by four IM injections of theHIV Protein vaccine, also given every other week, were well tolerated byNew Zealand White rabbits.

D. Exploratory DNA/PLG Local Irritation Tolerance Study in Male NewZealand White Rabbits—Single Dose Intramuscular

To assess the potential local irritant effects of various concentrationsof DNA/PLG in New Zealand White male rabbits when administered by asingle IM injection, the followings studies were performed using twogroups of 9 male rabbits each. On Day 1, each rabbit received a 0.5 mLIM injection of the test and control articles. Three rabbits/group werenecropsied one day (Day 2), one week (Day 8), or two weeks post-dosing(Day 15). Experimental design is depicted in Table 15.

Potential toxicity was evaluated based on clinical signs, dermal scoringof injection sites, body weight, comprehensive macroscopic examination,and microscopic evaluation of injection sites.

TABLE 15 Experimental Design Necropsy Day - No. of Treatment^(a) No. ofanimals Group Males IM Site 1 IM Site 2 IM Site 3 IM Site 4 2 8 15 1 9Saline 100 mg 1% DNA + 1% DNA + 3 3 3 PLG/PVA 100 mg 100 mg PLG (DF) PLG(RF) 2 9 0.1% 100 mg 2% DNA + 4% DNA + 3 3 3 CTBA PLG/PVA 50 mg 25 mgPLG (DF) PLG (DF) ^(a)Injection volume = 0.5 mL DF = DevelopmentFormulation RF = Research Formulation

There was no mortality and no treatment-related effects on body weight.Apparent bruising of the injection sites was observed sporadically in4/9 and 5/9 rabbits in Groups 1 and 2, respectively, during days 1-4.Bruising was noted at all injection sites except injection site # 2.This finding of slight bruising at the injection sites is consistentwith 1M injections. Results of the dermal Draize scoring of theinjection sites are presented in Table 16. Very slight edema was notedin two Group 1 rabbits (E sites 3 and 4) on Days 13 to 15 and in oneGroup 2 rabbit (IM site 4) on Days 13 to 14. Postmortem macroscopicfindings were limited to the injection sites and consisted of red firmareas, tan areas, hemorrhage on fascia overlying muscle, andsubcutaneous hemorrhagic areas. These findings were more prevalent onDay 2. Histopathological examination of the injection sites revealed thecharacteristic response to needle trauma (muscle fiber degeneration andhemorrhage) in the saline treated sites. Evaluation of the test articletreated sites revealed, on Day 2, minimal to mild treatment-relatedinflammation that was similar for all formulations. On Day 8,granulomatous changes were the predominant findings and there was nodifference between the formulations. These granulomatous changes areconsistent with know responses to PLG microspheres and/or theregenerative process. By Day 15, the histological changes were partially[1% DNA+100 mg PLG (development and research formulations), 2% DNA+50 mgPLG, 4% DNA+25 mg PLG] or fully resolved (100 mg PLG/PVA). See, alsoTable 16.

TABLE 16 Dermal Irritation Results Group Test/Control ArticleIdentification Findings 1 Saline None 100 mg PLG/PVA None 1% DNA + 100mg PLG (DF) Very slight edema in 1 rabbit on Days 13-15. 1% DNA + 100 mgPLG (RF) Very slight edema in 1 rabbit on Days 13-15. 2 0.1% CTAB None100 mg PLG/PVA None 2% DNA + 50 mg PLG (DF) None 4% DNA + 25 mg PLG (DF)Very slight edema in 1 rabbit on Days 13-14. DF = DevelopmentFormulation RF = Research Formulation

In conclusion, various concentrations of DNA/PLG were well toleratedwhen administered to male New Zealand White rabbits as a single IMinjection. Injection site findings were most frequent/strongest (mild tominimal) on day 2 and were partially to fully resolved by the end of therecovery period.

E. Multiple-Dose (Two) Mouse Immunogenicity Study with PCR InjectionSite Assessment

To assess immunogenicity and persistence of Gag-DNA PLG formulations atthe IM injection sites, ten female BALB/c mice per group were treated asoutlined in Table 17. Animals were dosed on days 0 and 28 and IMinjection sites were harvested 4 and 8 weeks post-last dose. Theformulations tested in this study were similar to the formulation usedin the toxicology studies.

TABLE 17 Experimental Design No of Necropsy - No of mice Group miceTreatment^(a) Main^(b) Recovery^(c) 1 10 1 μg Gag-DNA, 24 μg PLG 5 5 210 10 μg Gag-DNA, 240 μg PLG 5 5 3 10 10 μg Gag-DNA 5 5 ^(a)Administeredby IM injection on Days 0 and 28; ^(b)Four weeks post-last dose;^(c)Eight weeks post-last dose

Results of the PCR analysis of injection sites are presented in Table18. Results showed that the DNA-PLG formulations were comparable to thenaked-DNA control with regard to persistence. Although the Gag-DNA wasstill detectable at the injection site 4 and 8 weeks post-last dose, theamount remaining was insignificant (approximately 10⁻⁷% of the amount ofDNA injected).

TABLE 18 PCR Analysis of Injection Sites Group and Mean DNA StandardTreatment Time copy number^(c) Deviation % from Time 0 1 0 1.6 × 10¹¹ 0100 1 μg Gag-DNA, Main Necropsy^(a) 470.6 378.9 2.9 × 10⁻⁷ 24 μg PLGRecovery Necropsy^(b) 178.4 74.5 1.1 × 10⁻⁷ 2 0 1.6 × 10¹² 0 100 10 μgGag-DNA, Main Necropsy^(a) 1061.4 432.7 6.6 × 10⁻⁸ 240 μg PLG RecoveryNecropsy^(b) 209 108.0 1.3 × 10⁻⁸ 3 0 1.6 × 10¹² 0 100 10 μg Gag-DNAMain Necropsy^(a) 473 108.7 3.0 × 10⁻⁸ Recovery Necropsy^(b) 66.3 22.94.1 × 10⁻⁸ ^(a)Four weeks post-last dose ^(b)Eight weeks post-last dose^(c)The mean DNA copy number at time 0 was estimated based on the numberof copies/μg of DNA injected.

CONCLUSIONS

Under the conditions of these studies, single and/or multipleadministrations of the HIV vaccine formulation was well tolerated inanimal models (New Zealand White rabbits and BALB/c mice) and, inaddition, the formulations elicited potent immune responses. In themultiple-dose rabbit study, the HIV vaccine formulation produced notreatment-related adverse effects on clinical observations, body weightsand temperatures, food consumption, and clinical pathology (hematology,coagulation, and clinical chemistry). Dermal scoring of injection sitesrevealed occasional instances of very slight to slight erythema oredema, which appeared to be reversible. These findings at the injectionsite are consistent with those observed in a single-dose local tolerancerabbit study. In the latter, histopathological evaluation revealedtreatment-related minimal to mild inflammation at the injection site,which partially or fully resolved by the end of the recovery period. Infurther studies, PCR analysis of the injection sites demonstrated thatthe Env-DNA PLG did not integrate into the host genomic DNA and that theGag-DNA PLG did not persist at the injection sites after 4 or 8 weeks.

In the multiple-dose rabbit study, animals received the planned clinicaldose (1 mL HIV DNA vaccine, 0.5 mL HIV Protein vaccine/dose) by theclinical route of administration (IM). However, rabbits received fourdoses each of the HIV DNA vaccine and the HIV Protein vaccine, exceedingthe intended clinical regimen (three doses each) by one dose. Further,on a body weight basis, the dose in rabbits (approximately 2.5 Kg) wasapproximately 24 times higher than the same dose in humans(approximately 60 Kg). Therefore, administration of the clinical doseand regimen to normal human subjects is expected to be well tolerated.

In addition, the vaccine formulations were shown to be immunogenic ashigh titers of antibodies Gag and Env were observed.

Example 5 Enhanced Potency of Plasmid DNA/PLG Microparticle HIV Vaccinesin Rhesus Macaques Using a Prime-Boost Regimen with Recombinant Proteins

The following study was conducted to determine the effect ofPLG-mediated delivery on immunogencity.

A. Preparation of Vectors, Protein, PLG

HIV vaccines as described herein were evaluated in rhesus macaques asfollows. Plasmids pCMVKm2.GagMod.SF2 and pCMVKm2.o-gp140.SF162 wereprepared essentially as described in U.S. Pat. No. 6,602,705. Sindbisconstructs were prepared by excising the gag and env inserts frompCMVKm2 constructs and ligating them into pSINCP (a modified version ofpSIN1.5, as describe essentially in Hariharan et al. (1998) J Virol72(2):950-8).

Recombinant Env protein o-gp140SF162ΔV2 was produced in CHO cells andpurified essentially as described in Srivastava et al. (2003) J Virol.77(20):11244-11259.

Cationic PLG microparticles were prepared as follows. The microparticleswere prepared using an IKA homogenizer at high speed to emulsify 10 mlof a 5% w/v polymer solution in methylene chloride with 1 mL of PBS. Theprimary emulsion was then added to 50 ml of distilled water containingCTAB (0.5% w/v). This resulted in the formation of awater-in-oil-in-water emulsion that was stirred at 6000 rpm for 12 hoursat room temperature, allowing the methylene chloride to evaporate. Theresulting microparticles were washed four times in distilled water bycentrifugation at 10,000 g and freeze dried. The DNA was adsorbed ontoPLG-CTAB microparticles by incubating 1 mg of DNA in 1 ml of 1×TE bufferwith 100 mg of microparticles overnight at 4° C. with gentle rocking.The microparticles were then pelleted by centrifugation at 10,000 rpmfor 10 minutes, washed with 1×TE buffer, re-centrifuged, and suspendedin 5 ml of deionized water and freeze dried. The size distribution ofthe microparticles was determined using a particle size analyzer (Mastersizer, Malvern Instruments, UK).

DNA constructs were adsorbed onto PLG particles are described above.Similarly, HIV p55 gag protein was adsorbed onto anionic PLGmicroparticles as follows. Microparticles were prepared by homogenizing10 ml of 6% w/v polymer solution in methylene chloride with 40 ml ofdistilled water containing SDS (1% w/v) at high speed using a 10 mmprobe. This resulted in an oil-in-water emulsion, which was stirred at1000 rpm for 12 hours at room temperature, and the methylene chloridewas allowed to evaporate. The resulting microparticles were filteredthrough 38 μm mesh, washed 3 times in distilled water, and freeze-dried.The size distribution of the microparticles was determined using aparticles size analyzer (Master sizer, Malvem Instruments, UK).

50 mg of lyophilized SDS blank particles were incubated with 0.5 mg ofp55gag protein in 10 ml 25 mM Borate buffer, pH 9, with 6M Urea. 50 mglyophilized DSS blank microparticles were incubated with 0.5 mg of gp120protein in 10 mL PBS. Particles were left on a lab rocker, (Aliquotmixer, Miles Laboratories) at room temperature for 5 hours. Themicroparticles were separated from the incubation medium bycentrifugation, and the SDS pellet was washed once with Borate bufferwith 6 M urea, then three times with distilled water, and lyophilized.

The loading level of protein adsorbed to microparticles was determinedby dissolving 10 mg of the microparticles in 2 ml of 5% SDS-0.2 M sodiumhydroxide solution at room temperature. Protein concentration wasmeasured by BCA protein assay (Pierce Rockford, Ill.). The Zetapotential for both blank and adsorbed microparticles was measured usinga Malvern Zeta analyzer (Malvern Instruments, UK).

B. Vaccination

Rhesus immunization studies were undertaken to evaluate two DNA vaccinevectors and a cationic PLG microparticle DNA delivery system in aprime-boost regimen with recombinant proteins. Groups of 5 rhesusmacaques were immunized by intramuscular injection. injection on weeks0, 4 and 14 with DNA vaccines encoding HIV SF2 Gag (0.5 mg) and HIVSF162 gp140 Env (1.0 mg) with or without adsorption to PLGmicroparticles. The animals were boosted with yeast-derived p55 Gagprotein adsorbed onto anionic PLG microparticles (Gag/PLG) on week 29.Finally, the animals were boosted with CHO cell-derived oligomeric gp140Env protein with a deleted V2 region administered with the oil-in-waterMF59 adjuvant (Env/MF59) on weeks 38 and 75.

Immunogenicity of the vaccine compositions was assessed at various timesafter each immunization by quantitative and qualitative measurements ofantibody (ELISA, neutralization) and T cell responses(lymphoproliferation, intracellular cytokine staining, CTL).

C. Antibody Assays

The antibody responses against Env and Gag proteins were measured by anenzyme-linked immunosorbent assay (ELISA). For both ELISA's, NuncMaxisorp plates were coated overnight at 4° C. with 50 μl of 5 μg/ml ofEnv protein or Gag protein in PBS, pH 7.0. The coated wells were blockedfor 1 hr at 37° C. with 150 μl of 5% goat serum (Gibco BRL, GrandIsland, N.Y.) in phosphate-buffered saline (PBS). Serum samples wereinitially diluted 1:25 or 1:100 in the Blocking buffer followed bythree-fold serial dilution. The bound antibodies were detected withhorseradish peroxidase-conjugated goat anti-monkey IgG (SouthernBiotechnology Associates, Inc, diluted 1:5,000 with the blocking buffer)and incubated for 1 hour at 37° C. For development, 3,3′,5,5′tetramethylbenzidine (TMB) was incubated for 15 minutes according to themanufacturer's instructions, and the reaction was stopped by adding 2NHCL. The assay plates were then read on an ELISA plate reader at anabsorbance wavelength of 450 nm. A serum standard was included on eachmicrotiter plate, and a reference value of the standard was used for thenormalization of the sample ELISA titers. The titers represent theinverse of the serum dilution, giving an optical density of 0.5. Virusneutralizing antibodies were assessed against homologous HIV-1 SF162virus, using standard techniques.

D. Purification of Rhesus PBMC and Derivation of B Lymphoblastoid CellLines (B-LCL)

Rhesus peripheral blood mononuclear cells (PBMC) were separated fromheparinized whole blood on Ficoll-Hypaque gradients. To derive rhesusB-lymphoblastoid cell lines, PBMC were exposed to Herpesviruspapio-containing culture supernatant from the 594S cell line in thepresence of 0.5 μg/ml Cyclosporin A (Sigma). Rhesus PBMC were culturedat 2-3×10⁶ per well in 1.5 ml in 24-well plates for 8 days in AIM-V:RPMI1640 (50:50) culture medium (Gibco) supplemented with 10%heat-inactivated fetal bovine serum (AR10). Antigen-specific cells werestimulated by the addition of a pool of either gag or env peptides (10.7μg/ml total peptide). Recombinant human IL-7 (15 ng/ml, R&D Systems,Minneapolis, Minn.) was added at the initiation of culture. Human rIL2(Proleukin, 20 IU/ml, Chiron) was added on days 1, 3, and 6.

E. ⁵¹Cr-Release Assay for CTL Activity

Autologous B-LCL were infected with recombinant vaccinia viruses (rVV)expressing gag (rVVgag-pol_(SF2)) or env (rVVgp160env_(SF162)), thenlabeled overnight with Na₂[⁵¹Cr]O₄ (NEN, Boston, Mass.; 10 μCi per2.5×10⁵ B-LCL) and washed. Recombinant VV infected, ⁵¹ Cr-labeled B-LCLwere added (2500 per round bottom well) to duplicate wells containing3-fold serial dilutions of cultured PBMC. Unlabeled B-LCL (1×10⁵ perwell) were added to inhibit non-specific cytolysis. After 4 h, 50 μl ofculture supernatant was harvested, added to Lumaplates (Packard,Meriden, Conn.) and counted with a Wallac Microbeta TriLux liquidscintillation counter (Perkin Elmer Life Sciences, Boston, Mass.). ⁵¹Crreleased from lysed targets was normalized by the formula: Percentspecific ⁵¹Cr release=100%×(mean experimental release−spontaneousrelease)/(maximum release−spontaneous release), where spontaneousrelease=mean counts per minute (cpm) released from target cells in theabsence of PBMC and maximum release=mean cpm released from target cellsin the presence of 0.1% Triton X-100. A response was scored as positiveif the net specific lysis (antigen-specific minus non-specific lysis)was greater than or equal to 10% at two consecutive PBMC dilutions.

F. Lymphoproliferation Assay

2×10⁵ PBMC were incubated in flat bottom microtiter wells in a volume of0.2 ml AR10 in the absence or presence of p55 Gag protein (3 μg/ml) or apool of Env peptides (16 μg/ml). Six replicate cultures wereestablished. After 4 days incubation [³H]-thymidine ([³H]TdR, Amersham,Piscataway, N.J.) was added (1 μCi/well). Following overnightincubation, cultures were harvested onto glass microfiber filters.Cellular uptake of [³H]TdR was measured with a Microbeta TriLux liquidscintillation counter (Perkin Elmer).

G. Intracellular Cytokine Staining and Flow Cytometry

Rhesus PBMC were incubated overnight at 37° C. in the absence orpresence of antigen (gag peptide pool, 30 μg/ml, or env peptide pool, 30μg/ml). Anti-CD28 (1 μg/ml, Pharmingen, San Diego, Calif.) was added asa source of costimulation and Brefeldin A (1:1000, Pharmingen) was addedto prevent cytokine secretion. After overnight incubation PBMC werestained for cell surface CD4 (anti-CD4 allophycocyanin conjugate, cloneSK3, Becton Dickinson, San Jose, Calif.) and CD8 (anti-CD8a PerCPconjugate, clone SK1, Becton Dickinson), permeabilized withCytofix/Cytoperm (Pharmingen), and then stained for intracellular IFN-γ(monoclonal antibody 4S.B3, phycoerythrin conjugate, Pharmingen) andTNF-α (MAb11, FITC conjugate, Pharmingen). Stained cells were analyzedwith a FACSCalibur™ flow cytometer (Becton Dickinson).

H. Comparison of DNA Vaccine Vectors

Immunogenicity of DNA vectors without PLG was evaluated. For anti-Gagantibodies, neither vector (pCMV or pSINCP) was effective when given insaline as a primary immunization regimen. However, boosting of animalsprimed with naked gag DNA using Gag/PLG protein antigen rapidly inducedsignificant antibody responses. Similarly, Env/MF59 protein rapidlyboosted anti-Env antibodies. At no time was there a significantdifference in the antibody titers induced by pCMV or pSINCP.

Helper T cell responses were measured by both lymphoproliferation (LPA)and intracellular cytokine staining (ICS). Peripheral blood mononuclearcells (PBMC) were stimulated with recombinant p55gag protein or with apool of synthetic env peptides. As with antibody responses, the nakedpCMV and pSINCP DNA vaccines were not very effective at inducing LPA orICS responses. However, for Gag LPA responses, pSINCP seemed to begenerally more potent. Statistical significance between the pSINCP andpCMV groups was reached at weeks 20 and 27 (p=0.018, 0.023,respectively).

Similarly, pSINCP seemed to be more effective at inducing Env LPAresponses. Significantly higher LPA responses between groups wereobserved during DNA priming at weeks 20, 24, and 27 (p=0.028, 0.022, and0.044, respectively), as well as after the Env protein boost at week 44(p=0.016).

To quantify T cell responses further, PBMC were stimulated overnightwith antigen and then stained the PBMC with PE-conjugated anti-IFN-γ mAband FITC-conjugated anti-TNF-α mAb (intracellular). PBMC werecounterstained with APC-conjugated anti-CD4 and PerCP-conjugatedanti-CD8 and analyzed by flow cytometry for cytokine-positive cells,particularly for IFN-γ/TNF-α-double positive cells, which were the mostprevalent antigen-specific cells. No significant differences infrequencies of antigen-specific T cells were seen between groups ofanimals receiving pSINCP and pCMV.

For measurement of CTL, PBMC were cultured in the presence of a pool ofgag peptides or env peptides, IL-2, and IL-7. On day 8, PBMC cultureswere harvested, serially diluted, and added to microtiter wellscontaining ⁵¹ Cr-labeled autologous B-LCL that had been infected the daybefore with recombinant vaccinia vectors that expressed gag(rVVgagpol_(SF2)) or env (rVVgp160env_(SF162)). pCMV appeared morepotent at inducing Gag CTL responses than pSINCP, with a greater numberof responses over the course of the study. Neither DNA vaccine waseffective at inducing Env CTL.

In summary, both pCMV and pSINCP naked DNA vaccines induced antibody andT cell responses against HIV Gag and Env.

I. PLG Microparticle Delivery of DNA Vaccines

Animals were also immunized as described above with DNA/PLG compositionsto evaluate immunogenicity of DNA vaccines adsorbed to PLGmicroparticles. Adsorption of the HIV DNA vaccines onto cationic PLGmicroparticles was effective at enhancing immune responses, particularlyfor antibodies. PLG delivery markedly increased antibody titers inmacaques receiving either pCMV or pSINCP. During the DNA priming phase,anti-gag titers were significantly higher in the PLG groups compared tonaked DNA at every time point measured (p=0.0003 to 0.04), with peaktiters ˜1000-fold higher (FIG. 1). These differences were maintainedafter the protein boost, where pCMV/PLG and pSINCP/PLG were ˜10- to25-fold higher compared to naked DNA (p=0.02 to 0.04). Anti-Env antibodyresponses were also significantly higher in the PLG groups, but only atthe peak response after DNA priming (2 and 6 weeks post second DNA)(P=0.003 to 0.015). Thereafter and during protein boosting, the anti-envtiters were similar in all groups. For the PLG/DNA vaccine groups, peakantibody responses were observed after the second DNA immunization,whereas 3 immunizations were required for peak responses by naked DNA.

The PLG/DNA vaccines induced helper T cell responses against Gag andEnv, as measured by LPA and ICS. By LPA, the magnitudes of the responsesin the naked and PLG/DNA groups generally were similar, but when grouped(pCMV+pSINCP), PLG had significantly higher responses at 6 weeks for Gagand 16 weeks for Env, compared to naked DNA (p=0.05). The frequencies ofcytokine production by CD4 T cells, as measured by ICS, showed enhancedresponses in the Gag PLG group (pCMV+pSINCP groups combined) versusnaked DNA at 2 weeks post second DNA (p<0.05). No differences wereobserved for the Env DNA vaccines. CD8 T cells responses were measuredby ICS and ⁵¹Cr release. By ICS, the responses were generally low and nodifferences were seen among the groups. By ⁵¹ Cr release of culturedPBMC, good CTL responses were detected against Gag, but not against Env.The total number of Gag CTL responses was 24 in the PLG groups and 18 inthe naked DNA groups over the course of the study, with an apparentearlier onset of anti-Gag CTL in the pCMV/PLG group (3 of 5 animals at 2weeks post first DNA).

In summary, PLG delivery of HIV DNA vaccines was effective at inducingantibody and cellular immune responses. Moreover, PLG significantlyenhanced immunogenic responses as compared to naked DNA. Particularlystrong enhancement of antibody responses was observed for both the pCMVand pSINCP DNA vaccines. For Gag, this was true during both the DNApriming and protein boosting phases of the study. Cellular immuneresponses also were enhanced in some cases by PLG during DNA priming, asseen by earlier onset, increased magnitude, and increased frequency ofresponses.

J. Protein Boosting

The animals were boosted with recombinant Gag protein adsorbed ontoanionic PLG microparticles at 29 weeks, then with recombinant Env inMF59 adjuvant at 38 and 75 weeks (15, 24, and 51 weeks, respectively,after the last DNA immunization). Antibody titers were boosted markedlyin all groups (FIGS. 1,2). After boosting with gag protein the anti-gagantibody titers were approximately tenfold higher in the animals primedwith PLG/CTAB-DNA than those primed with naked DNA. The anti-gag titersequaled (DNA/PLG) or exceeded (DNA/saline) the peak titers achieved byDNA priming. For Env, titers in all groups were significantly boostedabove peak titers after DNA priming (p=0.0002 to 0.02) (FIG. 2). Thesecond Env protein boost restored antibody titers to levels seen afterthe first Env protein boost. Virus-neutralizing antibody responses werenot detected in any animals after DNA vaccine priming. However,increasing titers were observed after one and two protein boosterimmunizations, with overall geometric mean titers of 8 and 64,respectively (p=0.00071) (FIG. 3). At both of these time points, thetiters were not statistically different among the various vaccinegroups.

T cell responses also appeared to be boosted after protein immunization.For Gag, mean SI increased 4- to 7-fold over baseline after proteinboosting, with the number of responders increasing from 7 to 14 (out of20). However, the magnitude of the responses was not higher than thoseseen at the peak after DNA priming.

After Env protein boosting, mean SI increased 11- to 25-fold overbaseline and these responses were higher than those measured after DNApriming. By ICS, little or no increases were observed after Gag proteinboosting, but substantial increases in the proportion of cells secretingIFN-γ and TNF-α were seen after each Env protein boosting. Furthermore,the overall magnitude of the ICS response was higher after the secondcompared to the first protein boost (p=0.0008) (FIG. 4), with responsesapproaching 4% of CD4 T cells in some animals. As expected, CTLresponses were not boosted by protein immunization.

In summary, boosting DNA-primed macaques with recombinant Gag and Envproteins resulted in rapid and significant enhancement of antibody and Tcell responses. In some cases, the magnitude of these responses wasmarkedly higher than achieved after DNA priming.

Thus, DNA/PLG vaccines as described herein induce strong immuneresponses in rhesus macaques, with particular enhancement of antibodyresponses and an effect on helper and cytotoxic T cells. Theeffectiveness of boosting DNA/PLG-primed macaques with recombinantprotein was also established, including strong Th1-type cytokineproduction from T cells after Env protein boosting.

Example 6 Human Studies

Based on data from previous HIV vaccine trials (with other products),the rate of serious adverse experiences in the placebo controls isapproximately 3.5%. Extensive safety data on the use of otherrecombinant glycoprotein antigens with MF59 indicate that such vaccineantigens, when administered with MF59, are very safe and generally welltolerated. Additionally, these vaccines have elicited a strong antibodyresponse against the particular antigens.

An exemplary protocol for human studies is shown below in Table 19.Although exemplified with regard to subtype B, it will readily apparentthat the protocol can also be used as is, or with modifications, forother strains or subtypes of HIV.

TABLE 19 Human Protocol Immunization Schedule in Months (Days) DNAProtein 6 9 Group #/grp dose dose 0 (0) 1 (28) 2 (56) 4 (112) (168)(236) PART ONE 1 10 250/250 100 μg A A A B B 2 Placebo P P P P P 2 10500/500 100 μg A A A B B 2 Placebo P P P P P 3 10 1000/1000 100 μg A A AB B 2 Placebo P P P P P PART TWO 4 20 1000/1000 100 μg A A A B B 4Placebo P P P P P 5 30 1000/1000 100 μg A A A + B B 6 Placebo P P P P 630 1000/1000 100 μg A A A + B B 6 Placebo P P P P 7 30 1000/1000 100 μgA A B B 6 Placebo P P P P TOTAL: 168 STUDY AGENTS A: Clade B Gag + EnvDNA/PLG microparticles, dose indicated below (μg) B: Clade B gp140 Envprotein, 100 μg P: Placebo: PBS

1. An immunogenic composition comprising oligomeric gp140 (o-gp140) anda pharmaceutically acceptable excipient.
 2. The immunogenic compositionof claim 1, wherein the concentration of o-gp140 is between about 0.1and 10 mg/mL.
 3. The immunogenic composition of claim 1, wherein theconcentration of o-gp140 per dose is approximately 100 μg/dose.
 4. Theimmunogenic composition of claim 1, containing: (a) 0.4 mg/ml o-gp140,2.75 mg/ml sodium citrate dihydrate, 0.15 mg/ml citric acid monohydrate,17.53 mg/ml sodium chloride; 2 mg Env-DNA/ml, 50 mg PLG/ml, and 2 mgGag-DNA/ml; or (b) 0.4 mg/ml Env protein and MF59.
 5. The immunogeniccomposition of claim 1, further comprising an adjuvant.
 6. Theimmunogenic composition of claim 5, wherein the adjuvant is MF59 or CpG.7. The immunogenic composition of claim 6, wherein the adjuvant is MF59and MF59 comprises 39 mg/ml squalene, 4.7 mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.68 mg/ml sodium citrate dihydrate, 0.17mg/ml citric acid monohydrate.
 8. An immunogenic composition comprising:(a) an HIV Env DNA immunogenic composition, said HIV Env DNA immunogeniccomposition comprising at least one HIV Env encoding polynucleotidesequence and PLG; (b) an HIV Gag DNA immunogenic composition, said HIVGag DNA immunogenic composition comprising at least one HIV Gag-encodingpolynucleotide sequence and PLG: and (c) an HIV protein immunogeniccomposition, said HIV immunogenic composition comprising o-gp140 and apharmaceutically acceptable excipient.
 9. A method of generating animmune response in a subject, said method comprising: (a) administeringto the subject at least one immunogenic composition, said compositioncomprising: (i) a nucleic acid expression vector comprising at least oneHIV Gag- or Env-encoding polynucleotide sequence; or (ii) an HIV ogp140;and (b) administering to the subject, at a time subsequent to theadministering of step (a), at least one immunogenic composition, saidcomposition comprising: (i) a nucleic acid expression vector comprisingat least one HIV Gag- or Env-encoding polynucleotide sequence; or (ii)an HIV ogp140.
 10. A method of generating an immune response in asubject, said method comprising: (a) administering to said subject atleast one HIV DNA immunogenic composition comprising a nucleic acidexpression vector comprising at least one HIV Gag- or Env-encodingsequence; and (b) administering to the subject, at a time subsequent tothe administering of step (a), at least one immunogenic compositioncomprising HIV ogp140.
 11. The method of claim 10, wherein step (a)comprises multiple administrations of said at least one HIV DNAimmunogenic composition and step (b) comprises multiple administrationsof said at least one immunogenic composition comprising HIV ogp
 140. 12.The method of claim 11, wherein step (a) comprises two or threeadministrations at one month intervals; step (b) comprises two or threeadministrations at 1, 2 or 3 month intervals; and the time between theadministrations of step (a) and step (b) is 1 to 5 months.
 13. Themethod claim 10, wherein step (a) comprises administering at least oneHIV Gag DNA immunogenic composition and at least one HIV Env DNAimmunogenic composition.
 14. The method of claim 9 wherein step (b)comprises concurrently administering at least one DNA immunogeniccomposition comprising a nucleic acid expression vector comprising atleast one HIV Gag- or Env-encoding sequence and at least one HIVimmunogenic composition comprising ogp140.
 15. The method of claim 14,wherein step (a) comprises administering at least one HIV Gag DNAimmunogenic composition and at least one HIV Env DNA immunogeniccomposition.
 16. The method of claim 9, wherein at least oneadministration is intramuscular or intradermal.
 17. A method of makingoligomeric HIV Env gp140 proteins, comprising the steps of introducing anucleic acid encoding gp140 into a host cell; culturing the host cellunder conditions such that gp140 is expressed in the cell; and isolatingo-gp140 protein from the host cell.
 18. The method of claim 17, whereinthe o-gp140 is secreted from the cell and isolated from the cellsupernatant.
 19. A method of making a composition according to claim 1,comprising combining o-gp140 with an adjuvant.
 20. The immunogeniccomposition of claim 8, wherein the concentration of PLG is betweenabout 5 and 100 fold greater than the concentration of the nucleic acidexpression Vector.
 21. The immunogenic composition of claim 20, whereinthe concentration of nucleic acid is between about 10 μg/mL and 5 mg/mLand the concentration of the PLG is between about 100 μg/mL and 100mg/mL.
 22. The immunogenic composition of claim 8, wherein theconcentration of nucleic acid per dose is between approximately 1μg/dose and 5 mg/dose and the concentration of the PLG per dose isbetween about 10 μg/dose and 100 mg/dose.
 23. The immunogeniccomposition of claim 8, wherein the HIV Env DNA immunogenic compositioncomponent is an aqueous solution comprising 50.0 mg/ml PLG, 20 mg/mlplasmid DNA, 0.5 mg/ml hexadecyltrimethylammonium bromide, 44 mg/mlmannitol, 14.7 mg/ml sucrose, 0.37 mg/ml EDTA, 1.4 mg/ml sodium citratedihydrate, and 0.04 mg/ml citric acid monohydrate; or comprises: 10 μgDNA and 0.25 mg PLG; 100 μg DNA and 2.5 mg PLG; 10 μg DNA and 9.25 mgPLG; or 100 μg DNA and 2.5 mg PLG.
 24. The immunogenic composition ofclaim 8, wherein the HIV Gag DNA immunogenic composition component is anaqueous solution comprising 50.0 mg/ml PLG, 2.0 mg/ml plasmid DNA, 0.5mg/ml hexadecyltrimethylammonium bromide, 44 mg/ml mannitol, 14.7 mg/mlsucrose, 0.37 mg/ml EDTA, 1.4 mg/ml sodium citrate dihydrate, and 0.04mg/ml citric acid monohydrate; or comprises: 10 μg DNA and 0.25 mg PLG;100 μg DNA and 2.5 mg PLG; 10 μg DNA and 9.25 mg PLG; or 100 μg DNA and2.5 mg PLG.
 25. The immunogenic composition of claim 8, wherein theconcentration of o-gp140 is between about 0.1 and 10 mg/mL.
 26. Theimmunogenic composition of claim 8, wherein the concentration of o-gp140per dose is approximately 100 μg/dose.
 27. The immunogenic compositionof claim 8, wherein the HIV immunogenic composition component contains:0.4 mg/ml o-gp140, 2.75 mg/ml sodium citrate dihydrate, 0.15 mg/mlcitric acid monohydrate, 17.53 mg/ml sodium chloride; 2 mg Env-DNA/ml,50 mg PLG/ml, and 2 mg Gag-DNA/ml; or 0.4 mg/ml Env protein and MF59.28. The immunogenic composition of claim 8, wherein the HIV proteinimmunogenic composition component further comprises an adjuvant.
 29. Theimmunogenic composition of claim 28, wherein the adjuvant is MF59 orCpG.
 30. The immunogenic composition of claim 29, wherein the adjuvantis MF59 and MF59 comprises 39 mg/ml squalene, 4.7 mg/ml polysorbate 80,4.7 mg/ml sorbitan trioleate 2.68 mg/ml sodium citrate dihydrate, 0.17mg/ml citric acid monohydrate.
 31. The method of claim 10, wherein atleast one administration is intramuscular or intradermal.
 32. An HIVimmunogenic composition comprising a nucleic acid expression vectorcomprising at least one HIV Gag- or Env-encoding sequence; and PLG.